P104 PRECLINICAL TRIAL OF A GSK3 AND PDE7 DOUBLE INHIBITOR IN SOD1G93A MICE: A GENDER SPECIFIC EFFECT
ATENCIA G1, GIL C2, PALOMO V2, CALVO AC3, JUAREZ-RUFIÁN A1, MARTÍN-CASANUEVA MÁ4, CORDERO-VÁZQUEZ P1, MUÑOZ-BLANCO JL5, ESTEBAN-PÉREZ J1, OSTA R3, MARTÍNEZ A2, GARCÍA-REDONDO A1
1Neurology Department – ALS Unit, CIBERER U-723. Health Research Institute, October 12th University Hospital, Madrid, Spain, 2Medical Chemistry Institute. CSIC, Madrid, Spain, 3LAGENBIO-I3A – Veterinary Faculty of Zaragoza – Aragonese Institute of Health Sciences (IACS), University of Zaragoza, Spain, 4Biochemistry Department, CIBERER U-723. Health research Institute, October 12th University Hospital, Madrid, Spain, 5Neurology Department – ALS Unit, Gregorio Marañón University Hospital, Madrid, Spain
Email address for correspondence: [email protected]
Keywords: preclinical assay, GSK-3, PDE-7
Background: Glycogen synthase kinase (GSK)-3 has recently been implicated in the pathogenesis of neurodegenerative diseases. This enzyme is currently used as a neuroprotector agent and it modulates the apoptotic pathway because the inhibition of GSK-3 stops the intrinsic apoptosis (1).
cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDE), then these enzymes modulate cyclic nucleotide-mediated signal pathway like neuroinflammatory responses (2,3). From the different phosphodiesterases expressed, PDE-7 is expressed in T-cells, brain and skeletal muscles.
The GSK-3 and PDE7 inhibition could prevent motor neuron apoptosis, increase levels of cAMP in muscle of ALS patients and repress neuroinflammatory symptoms.
VP1.15 was originally synthesized as an inhibitor of GSK-3 but also presents inhibitory capacity for PDE7 (with IC = 2 microM and IC = 1.11 microM respectively).
Objectives: The aim of this project was to prove VP1.15 in the transgenic hSOD1 mutant mice developing a preclinical assay.
Methods: A total of 30 transgenic female mice and 22 transgenic male mice were recruited (all of them tested for the human transgene). In a previous study in wild type mice we determined that a dose of 4 mg/kg was not toxic.
The trial began at the time of onset of symptoms (87.5 ± 2.3 days of life) that were analyzed by taking the weight three times a week, by visual assessment of motor neuron impairment and by testing grid once a week. The previous selected dose was administrated by intraperitoneal injection during the following 35 days.
Results: Medium survival: Males - untreated 127/treated 138.5 days; Females - untreated 142/treated 144 days. Kaplan-Meier survival analysis gives the following values of p all animals: p = 0.7403 (females); p = 0.0774 (males); values obtained from littermates: p = 0.8027 (females); p = 0.0667 (males).
There are not obvious differences in the evolution of weight in treated and untreated males. However, treated females have less weight loss compared to untreated.
No differences were found when evaluating the neuromuscular deterioration between treated and untreated females. By contrast, neuromotor involution of untreated males is faster than treated males. Treated males exhibit a progressive loss of neuromuscular skills to rates similar to females. Similar findings were obtained in grill test.
Discussion and conclusions: Males respond to the treatment improving their survival about 10 days and no effect was apparently observed in females. The big differences between sexes could be related with the inhibition of VP1.15 that simulates the protective effect of estrogens, which was proven in this model (4). Subsequent studies are necessary to test the positive effect of VP1.15 in an ALS model in which senescent females develop the disease.
Funding: This study has been supported by ISCIII (PI071283 and EC08/00049) and by Agencia Laín Entralgo (NDG07/07).
References
- Koh SH, Kim Y, Kim HY, et al. Exp Neurol 2007; 205(2):336–46.
- Staines DR, Brenu EW, Marshall-Gradisnik S. Neuropsychiatr Dis Treat 2009;5:81–9.
- Mishra SK, Menon NK, Roman D, et al. J Neurol Sci 1992; 109(2):215–8.
- Choi CI, Lee YD, Gwag BJ, et al. J Neurol Sci 2008; 268 (1–2):40–7.
P105 RESCUE OF ADULT MOTONEURONES BY MECHANO-GROWTH FACTOR (MGF), LIVER-TYPE INSULIN-LIKE GROWTH FACTOR -1 (IGF-1EA) AND GLIAL CELL DERIVED NEUROTROPHIC FACTOR (GDNF) DELIVERED AT THE TIME OF NERVE INJURY
JOHNSON I, MAHADEVAN J, TURNBULL G
University of Adelaide, Adelaide, Australia
Email address for correspondence: [email protected]
Keywords: neurotrophic factors, adult, motoneurone
While much is known of the neurotrophic rescue of immature motoneurones, little is known about their rescue in the adult animal. We previously found that intramuscular transfer of the gene for a muscle-derived isoform of insulin-like growth factor-1 (MGF) one week before facial nerve avulsion rescued 80% of adult rat motoneurones when 80% would have died, and that it was twice as effective than the commonly- used liver-type isoform of IGF-1 (1). In this study we have examined the neuroprotective potential of MGF and IGF-1 peptides delivered at the time of facial nerve injury in adult rats and compared this with a known potent rescue factor for immature rat facial motoneurones, GDNF.
The facial nerve of groups of 4-8 anaesthetised 3m Sprague-Dawley rats was avulsed and 10μl of 1μg/μl of either MGF, liver-type IGF-1 or GDNF injected into the stylomastoid foramen. Control animals received 10μl of saline after avulsion, avulsion only or were non-operated. After 7 days, numbers of motoneurones were determined stereologically. Two additional groups of rats were also examined 14d and 28d after MGF injection.
Seven days following avulsion only, or avulsion + saline, 32% of adult motoneurones were lost. Injection of MGF, liver-type IGF-1 or GDNF immediately after avulsion reduced the motoneuronal loss to 19-21% (p < 0.05 vs. controls). Motoneuronal loss increased to approximately 50% 14-28d after MGF injection, while 80% were lost 28d following avulsion alone.
We show that MGF peptide delivered at the time of nerve injury has a similar neuroprotective effect to IGF-1Ea for mature motoneurones at 7 days. Rather than conferring absolute rescue, however, a single dose of MGF peptide reduces the rate of motoneuronal loss up to 28 days. We do not know if the neuroprotective for IGF-1Ec is maintained beyond 7 days, although intramuscular gene transfer of IGF1Ea rescues approximately 50% of adult rat facial motoneurones at 1 month (1). In contrast to intramuscular gene-transfer, injection of adenoviral vectors carrying genes for IGF-1 into the styolomastoid foramen at the time of adult rat facial nerve avulsion is not associated with motoneuronal rescue at 1 month (2).
Taken together, these results indicate that the mode of neurotrophic factor delivery and the post-operative time period examined in experimental models of motoneuronal degeneration can significantly affect the extent of neurotrophic rescue observed.
References
- Aperghis M, Johnson I, Cannon J, et al. Brain Res 2004; 1009:213–8.
- Sakamoto T, Kawazoe Y, Shen J-S, et al. J Neurosci Res 2003;72:54–64.
P106 MELITTIN EFFECTS IN AN ANIMAL MODEL OF ALS
YANG EJ, CHUNG SY, CHOI S-M
KIOM, Daejeon, Republic of Korea
Email address for correspondence: [email protected]
Keywords: hSOD1G93A, melittin
Amyotrophic lateral sclerosis (ALS) is a paralyzing disorder characterized by the progressive degeneration and death of motor neurons and occurs both as a sporadic and familial disease. Mutant SOD1 (mtSOD1) in motor neurons induces vulnerability to the disease through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport- and growth factor signaling, excitotoxicity, and neuro-inflammation.
Melittin is a 26 amino acid protein and is one of the components of bee venom which is used in traditional Chinese medicine to inhibit cancer cell proliferation and is known to have anti-inflammatory and anti-arthritic effects.
The purpose of the present study was to determine if melittin could suppress motor neuron loss and protein misfolding in the hSOD1G93A mouse, which is commonly used as a model for inherited ALS. Meltittin was injected at the ‘ZuSanLi’ (ST36) acupuncture point in the hSOD1G93A animal model. Melittin-treated animals showed a decrease in the number of microglia and in the expression level of phospho-p38 in the spinal cord and brainstem. Interestingly, melittin treatment in symptomatic ALS animals improved motor function and reduced the level of neuron death in the spinal cord when compared to the control group. Furthermore, we found an increased of a-synuclein modifications, such as phosphorylation or nitration, in both the brainstem and spinal cord in hSOD1G93A mice. However, melittin treatment reduced a-synuclein misfolding and restored the proteasomal activity in the brainstem and spinal cord of symptomatic hSOD1G93A transgenic mice.
Our research suggests a potential functional link between melittin and the inhibition of neuroinflammation in an ALS animal model.
P107 OLESOXIME (TRO19622) DELAYS MUSCLE DENERVATION, ASTROGLIOSIS, MICROGLIAL ACTIVATION AND MOTONEURON DEATH IN THE MURINE ALS MODEL SOD1G93A
SUNYACH C1, ARNOUX T2, MICHAUD M2, BERNARD N1, AEBISCHER J1, RAOUL C1, PRUSS R2, BORDET T2, PETTMANN B1
1Inserm-Avenir team, The Mediterranean Institute of Neurobiology, Inmed, Marseille, France, 2TROPHOS, Marseille, France
Email address for correspondence: [email protected]
Keywords: olesoxime, therapy, SOD1G93A
Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative pathology and riluzole, the only approved treatment, delays death by only a few months. The disease is mimicked to a striking degree in transgenic mice carrying human ALS-linked SOD1 gene mutations. These mice have therefore been extensively used to test potential therapeutic molecules. Olesoxime (TRO19622), a novel neuroprotective and reparative compound identified in a high-throughput screen based on motoneuron survival, delays disease onset and improves survival in mutant SOD1G93A mice, a model for ALS (1).
Objectives: The present study further analyses the cellular basis for the protection provided by olesoxime at the neuromuscular junctions (NMJ) and the spinal cord.
Methods: Studies were carried out at two disease stages, 60 days, presymptomatic and 100 days, symptomatic. Cohorts of wildtype and SOD1G93A mice were randomized to receive placebo or olesoxime-charged food pellets from day 21 onward. Body weight was recorded weekly. At 60 days, some mice were sacrificed to measure olesoxime levels in blood, brain and spinal cord. The others were anesthetized and their gastrocnemius muscles dissected out. After post-fixation, cryostat sections were analysed for NMJ integrity by double staining for a-bungarotoxin and neurofilament. At day 100, the same procedure as at day 60 was applied with in addition fixative perfusion after taking out the gastrocnemius and dissection of the diaphragm as well as of the spinal cord. Whole-mount immunostaining was used to study the diaphragm innervation and cryostat sections of the spinal cord were immunostained for VAChT to visualize motoneurons, GFAP for astrocytes and Iba-1 for microglial cells.
Results: Analysis at 60 days showed that olesoxime greatly protects the gastrocnemius muscle from denervation, reducing denervation from 60 to 30% compared to SOD1G93A mice fed with placebo food pellets (n = 5 mice/group). At the symptomatic stage, a preliminary analysis suggests that only a few NMJs were still preserved by olesoxime treatment both in gastrocnemius muscle and in the diaphragm. In the spinal cord, olesoxime strongly reduced astrogliosis and microglial activation and moderately prevented motoneuron loss (n = 3 mice/group). In addition, time of onset defined as the time mice started to loose weight was slightly delayed by olesoxime. Additional mice are currently being evaluated at 100 days to further confirm these results.
Discussion and conclusion: Though still incomplete, these studies suggest that olesoxime exerts its protective effect on multiple cell types implicated in the disease process in SOD1 mice, slowing down muscle denervation, astrogliosis, microglial activation and motoneuron death. A Phase 3 clinical study in ALS patients will determine whether olesoxime could be beneficial for the treatment of ALS.
Reference
- Bordet, et al. J. Pharmacol. Exp. Ther., 2007.
P108 ABERRANT NEUREGULIN1 SIGNALING IN AMYOTROPHIC LATERAL SCLEROSIS
SONG F1, CHIANG P1, RAVITS J2, LOEB J1
1Hiller ALS Clinic and Research Center, Department of Neurology, Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA, 2Translational Research Program Benaroya Research Institute, Seattle, WA, USA
Email address for correspondence: [email protected]
Keywords: neuregulin, microglia, motor neuron
Background: Neuregulin1 (NRG1) is a neuronally-expressed factor that supports axoglial and neuromuscular development through a number of alternatively spliced isoforms, but has not been studied in the pathogenesis and progression of amyotrophic lateral sclerosis (ALS). NRG1 is an important molecule for axoglial communication in the central nervous system and may be a new potential therapeutic target for patients with ALS.
Objectives: Here, we analyzed the relationship of NRG1 isoform expression with glial cell activation and motor neuron loss in the spinal cords of ALS patients as well as during disease progression in the ALS – superoxide dismutase 1 (SOD1) mouse model.
Methods: Pathological changes were measured for motor neuron number, demyelination, gliosis and microglial activation in both ALS patients and ALS-SOD1 (G93A) mice. Gene expression and protein levels were determined by quantitative PCR for a number of genes implicated in the disease process as well as the NRG1 gene isoforms that have important roles in axoglial interaction.
Results: Significant pathological changes that could be mediated by NRG1 signaling, including microgliosis, astrocytosis and motor neuron loss, were observed in the ventral horn spinal cord in both ALS patients and increased in SOD1 mice, as a function of disease progression. Whereas type III (membrane-bound) NRG1 expression was reduced in parallel with motor neuron loss, type I (secreted) NRG1 increased and was associated with glial activation. Consistently, excessive NRG1 receptor activation was observed on activated microglia in both ALS patients and in SOD1 mice. This activation was observed prior to upregulation of NRG1 gene expression, at the time of disease onset.
Discussion and conclusions: While the downregulation of membrane-bound type III NRG1 forms may be a marker of motor neuron loss, increased signaling by secreted type I NRG1 forms could contribute to disease pathogenesis through glial cell activation and could therefore represent a novel therapeutic target against disease progression in ALS.
P109 IMPAIRED NEUROPROTECTIVE RESPONSE OF MICROGLIA TO ACUTE NEURON INJURY AT THE DISEASE PROGRESSION STAGE IN ALS MODEL
KAWAMURA M, YAMASAKI R, KAWAMURA N, TATEISHI T, OHYAGI Y, KIRA J-I
Department of Neurology, Neurological Institute, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan
Email address for correspondence: [email protected]
Keywords: mSOD1, microglia, axotomy
Background: Dominant mutations in superoxide dismutase (mSOD1) cause a familial form of amyotrophic lateral sclerosis (ALS). Accumulating evidence indicates the occurrence of non-cell-autonomous motor neuron death in ALS models based on mSOD1. Activated microglia are seen in both human ALS and animal ALS models. Selective lowering of mSOD1 levels in microglia prolongs survival of mSOD1-Tg mice. Microglia play a significant role in disease progression; however, whether microglia are neurotoxic or neuroprotective remains undetermined. Motor nerve axotomy induces acute motor neuron damage and subsequent microglial responses.
Objectives: We aimed to elucidate whether microglial function is neurotoxic or neuroprotective to motor neurons in an ALS model, by studying temporal changes in microglial responses to acute motor neuron injury in mSOD1-Tg mice.
Methods: We performed unilateral hypoglossal nerve axotomy in mSOD1 (G93A) Tg mice in the presymptomatic stage (8 weeks of age) and the disease progression stage (17 weeks) and their non-transgenic littermates (NTG). On days 3 and 21 after axotomy the medulla, including the hypoglossal nucleus, was removed and sectioned, Nissl-stained for neuronal counting and immunostained with Iba1 for microglial counting. The numbers of neurons and microglia were counted. We also immunohistochemically examined the expression of neurotrophic factors including GDNF and IGF-1.
Results: Hypoglossal nerve axotomy resulted in recruitment of microglia and their encircling of hypoglossal motor neurons at three days after axotomy, which was followed by neuronal loss at 21 days after axotomy. There was no significant difference in the survival rates of motor neurons between mSOD1-Tg and NTG mice at three weeks after axotomy during the presymptomatic stage. By contrast, the survival rate of neurons in mSOD1-Tg mice was significantly lower than that in NTG mice after axotomy during the disease progression stage. There was no difference in the numbers of neurons between axotomized and non-axotomized sides at three days after axotomy. The number of microglia was significantly lower in mSOD1-Tg mice than in NTG mice during the disease progression stage, but no difference was seen during the presymptomatic stage. Activated microglia of mSOD1-Tg and NTG mice expressed neurotrophic factors including GDNF and IGF-1 during both the presymptomatic and disease progression stages.
Discussion and conclusion: In this study, we found that mSOD1 attenuates microglial responses to acute motor nerve injury during the disease progression stage, but not during the presymptomatic stage. Our findings indicate that reactive microglia are potentially neuroprotective after axotomy. Microglia are thought to heterogeneously include neurotoxic M1 and neuroprotective M2 phenotypes. Impaired neuroprotection from microglia may partly underlie ALS progression. The activation of neuroprotective microglia and normalization of cross talk between neurons and microglia during the disease progression stage may be a target of novel therapies for ALS.
P110 EVIDENCE FOR INVOLVEMENT OF THE INNATE IMMUNE TOLL-LIKE RECEPTOR SYSTEM IN THE SOD1G93A TRANSGENIC MOUSE MODEL OF MOTOR NEURON DISEASE
LEE JYP1, LEE JD1, TAYLOR SM1, PHIPPS S1, NOAKES PG1,2, WOODRUFF T1
1School of Biomedical Sciences; 2Queensland Brain Institute, The University of Queensland, Queensland, Brisbane, Australia
Email address for correspondence: [email protected]
Keywords: Toll-like receptors, SOD1G93A mice, neuroinflammation
Background: There is increasing evidence for a role of innate immune system components, such as the complement cascade and Toll-like receptors (TLRs), in the pathogenesis of motor neuron disease. Upregulation of numerous complement factors has previously been shown in mouse models of motor neuron disease. There is also evidence for a role of the TLRs in motor neuron disease; mRNA for several TLRs was shown in post-mortem brain tissue from human motor neuron disease patients, as well as a preliminary report of TLR expression in the mouse SOD1G93A transgenic model of motor neuron disease.
Objectives: The present study aimed to fully characterise the expression of several TLRs (TLR 2, 3, 4 and 7), and one of their endogenous ligands (high mobility group box 1; HMGB1) in SOD1G93A mice.
Methods: Expression of the above factors was compared across various pre-defined stages of the disease (pre- symptomatic, P28; onset stage, P65; mid-onset stage, P115; and end-stage, P175) in the lumbar spinal cord of C57BL6/ J SOD1G93A transgenic mice (n = 9), and their wild-type littermates (n = 9) using quantitative polymerase chain reaction (qPCR). Localisation of TLR2 and TLR4 in the lumbar spinal cord of end stage SOD1G93A mice was also investigated using immunohistochemistry.
Results: mRNA expression of TLR 2, 3, 4 and 7 and HMGB1 was significantly increased in SOD1G93A mice compared to wild-type mice at the end-stage of the disease. TLR3 and HMGB1 had a two-fold increase, TLR4 a five-fold increase, TLR2 a seven-fold increase, and TLR7 an eight-fold increase in mRNA expression in SOD1G93A mice compared to their wild-type littermates. TLR 3, 4, 7 and HMGB1 had no apparent change at mid-onset stage. By contrast, TLR2 expression doubled at the mid-onset stage in SOD1G93A mice. Immunohistochemistry demonstrated that TLR4 was mainly localised to astrocytes, and TLR2 to microglia, at end stage of disease.
Discussion and conclusions: These results suggest that the secondary effect of activating TLR pathways may contribute to the inflammatory progression in this SOD1G93A transgenic mouse model of motor neuron disease. Thus, further understanding the role of TLR activation in neuroinflammation is warranted, and may provide a potential novel therapeutic approach for treating motor neuron disease.
P111 UPREGULATION OF COMPLEMENT COMPONENTS IN MOTOR NEURON DISEASE
LEE J1, WOODRUFF T1, TAYLOR S1, NOAKES P1,2
1School of Biomedical Sciences; 2Queensland Brain Institute, the University of Queensland, St Lucia, QLD, Australia
Email address for correspondence: [email protected]
Keywords: neuroinflammation, complement system, CD88
Background: There is increasing evidence that neuroinflammation drives progression of many neurodegenerative diseases. The complement system, which is part of innate immune system, has recently been implicated in the pathogenesis of motor neuron disease (MND). Our previous studies in the hSOD1G93A rat model of MND demonstrated that excessive complement activation in the lumbar spinal cord contributed to motor neuron death. We have shown that hSOD1G93A rats treated with the selective C5a receptor (CD88) antagonist PMX205 had reduced gliosis and improvements in behavioral deficits, consistent with reduced neuropathology.
Objectives: The current study aimed to determine the expression and localization of C5a receptors, CD88 and C5L2 at both the mRNA and protein levels in the hSOD1G93A mouse model of MND and in MND patients.
Methods: Lumbar spinal cord from high-copy number C57BL/6J hSOD1G93A mice and their wild-type (WT) littermates were obtained at 4 different ages during disease progression, and expression and localization of CD88 and C5L2 were examined using qPCR, in situ hybridization, Western blotting and immuno-histochemistry. Circulating levels of C5a were also examined using an ELISA. Expression of CD88 in the motor cortex of MND patients were investigated using immunohistochemistry.
Results: We found consistent upregulation of plasma C5a levels which correlated with disease progression, and also upregulation of CD88 and C5L2 mRNA and protein levels in the lumbar spinal cord during disease progression. Immuno-localization showed that CD88 is expressed predominantly on the microglia surrounding the regions of motor neuron death. By contrast, the alternative C5a receptor C5L2 is expressed predominantly on astrocytes. CD88 was also upregulated in the motor cortex of MND patients when compared to normal patients.
Discussion and conclusions: These results indicate that complement activation, leading to increased expression of C5a and its receptors in the hSOD1G93A mice and MND patients has an important role in motor neuron death and may therefore play a role in the progression of MND. Hence reducing complement-induced inflammation could be an important therapeutic strategy to treat MND.
P112 PRE-SYMPTOMATIC NEUROINFLAMMATION IN ALS: A NOVEL METHOD FOR STUDYING TRANSCRIPTIONAL CHANGES IN MURINE VASCULAR ENDOTHELIUM
EVANS M1, STOLP H2,3, TALBOT K3, ANTHONY D2, SIBSON N4, TURNER M1
1Department of Clinical Neurology; 2Department of Pharmacology; 3Department of Physiology, Anatomy and Genetics; 4Gray Institute for Radiobiology, Oncology and Biology, University of Oxford, Oxford, UK
Email address for correspondence: [email protected]
Keywords: endothelium, neuroinflammation, SOD1
Background: Multiple sources of evidence have implicated inflammatory processes in the pathogenesis of ALS. Expression of the mutant protein in neurons or glia alone is insufficient to produce full pathology in the transgenic SOD1 mouse model of ALS, suggesting that the pathogenesis is non-cell autonomous. Haematogenous leucocytes may gain entry to the CNS and induce microglia to adopt either a pathogenic (M1) or protective (M2) phenotype. The changes that occur at the level of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) that allow leucocytes or inflammatory molecules to enter the CNS, might be a modifiable process in the treatment of human ALS.
Objectives: Through the novel application of an optimized protocol for endothelial cell isolation, we sought to investigate transcriptional changes in an experimental inflammatory model, and in pre-symptomatic transgenic SOD1 mice.
Methods: A single cell suspension of mouse neural tissue was added to a rabbit CD31 (PECAM) antibody-magnetic bead mixture. Endothelial cells were isolated by exposure to a magnet and a sample of endothelial-depleted and endothelial-enriched brain underwent FACS analysis.
For the inflammatory experiment, mice were injected into the left striatum with either saline, IL-1b (100 ng) or TNF-a (750 ng). RNA was extracted from the endothelial cells removed as described, converted to cDNA, and qPCR performed for the transcripts: IL1b, VCAM-1, ICAM-1, Claudin-5, TLR4 and aV integrin.
The sensorimotor cortex, brainstem, cervical enlargement and lumbar enlargement from 5 female SOD1G93A and 5 control littermates (80 days ± 1) were dissected, and endothelial cells extracted. RNA was extracted and converted to cDNA, and PCR performed for the same transcripts.
Results: FACS analysis confirmed isolation of endothelial cells with high purity (> 97% CD31+). Cell viability was very high, with < 1% of cells positive for propidium iodide. From the model of experimental inflammation endothelial cells from both injected groups showed higher levels of IL-1b, VCAM-1 and ICAM-1, and lower levels of Claudin-5 compared to saline-injected mice. In the SOD1 model expression of VCAM-1 and ICAM-1 was increased in the cortex, brainstem and cervical spinal cord, and TLR4 and aV integrin in the cortex, compared to controls.
Discussion: It was possible to differentiate cytokine-injected from control animals using RT-PCR of the endothelial cell RNA, and to demonstrate inflammation-related transcriptional alterations in the vascular endothelium in pre-symptomatic SOD1 mice.
Conclusions: Understanding the earliest pathological changes in models of ALS holds promise for developing novel disease biomarkers and drug targets in the human disease.
P113 DYSREGULATED PERIPHERAL IMMUNE RESPONSE EITHER PROMOTES MOTONEURON DEATH OR FAILS TO SUPPORT MOTONEURON SURVIVAL IN AN ALS MOUSE MODEL
MESNARD N1,2, HAULCOMB M3, XIN J1,2, TANZER L1,2, SANDERS V4, JONES K3
1Loyola University Chicago, Maywood, IL, USA, 2Hines VA Hospital, Hines, IL, USA, 3Indiana University, Indianapolis, IN, USA, 4The Ohio State University, Columbus, OH, USA
Email address for correspondence: [email protected]
Keywords: SOD1, T cells, axotomy
Background: Amyotrophic lateral sclerosis (ALS) axonal pathology and pre-synaptic deafferentation precede motoneuron (MN) degeneration during disease progression in patients and in an ALS mouse model (mSOD1). Previously in our laboratory, the phenotypic molecular response of wild-type (WT) and pre-symptomatic mSOD1 facial MN (FMN) and surrounding neuropil following facial nerve transection was characterized in a laser microdissection study. The MN gene expression response to axotomy is similar between WT and pre-symptomatic mSOD1 mice geared towards survival and axonal regeneration. However, the non-neuronal cells surrounding mSOD1 MN exhibit a differential expression pattern after axotomy compared to WT, potentially contributing to abnormal neuronal-neuropil cellular interactions to cause the increased MN degeneration. Since we observed a dysregulated microenvironment surrounding MN cell bodies centrally and previously showed that CD4 + T cells are required to maintain WT levels of MN survival and nerve regeneration, we proposed that the microenvironment surrounding the MN axons peripherally may also be dysregulated.
Objective: The present study investigated the neuroprotective functionality of mSOD1 peripheral immune cells in their ability to promote facial nerve regeneration and FMN survival levels.
Methods: First, WT and pre-symptomatic mSOD1 mice received a facial nerve crush axotomy to determine facial nerve regeneration via behavioral functional recovery assessments. All mice were observed daily until complete functional recovery was observed for eye blink reflex, vibrissae orientation, and vibrissae movement. FMN survival levels were assessed at 28 days post-axotomy. Secondly, several immunoreconstitution experiments were conducted using the adoptive transfer of either whole splenocytes or CD4 + T cells isolated from the whole splenocytes into immunodeficient (RAG2 KO) or pre-symptomatic mSOD1 mice. The adoptively transferred cells were injected into the tail vein of the mice 1 week prior to facial nerve transection. The adoptive transfer groups include RAG2 KO mice + mSOD1 splenocytes, mSOD1 mice + WT splenocytes, RAG2 KO mice + WT CD4 + T cells, RAG2 KO mice + mSOD1 CD4 + T cells, and mSOD1 mice + WT CD4 + T cells. FMN survival levels were assessed at 28 days post-axotomy.
Results: Pre-symptomatic mSOD1 mice demonstrated a delayed functional recovery response compared to WT after a facial nerve crush. Unlike WT splenocytes, pre-symptomatic mSOD1 splenocytes are not capable of neuroprotection to rescue the axotomy-induced cell death in immunodeficient mice. However, mSOD1 CD4 + T cells, isolated from the diseased/inflammatory microenvironment and placed in a non-diseased microenvironment, are capable of mediating neuroprotection to a similar level as WT CD4 + T cells.
Conclusions: The peripheral immune response in pre- symptomatic mSOD1 mice resembles that observed in immunodeficient mice following axotomy. Furthermore, the pre-symptomatic mSOD1 peripheral immune microenvironment prevents the activation of neuroprotective CD4+T cells.
Support: NIH grant NS40433 (KJJ & VMS)
P114 PROGRESSIVE ACCUMULATION OF NERVE/GLIAL ANTIGEN 2 PROTEOGLYCAN IN THE SPINAL CORD WITH MUTANT SOD1-INDUCED NEURODEGENERATION IN RATS
WARITA H1, MIZUNO H1, SUZUKI N1, ITOYAMA Y2, AOKI M1
1Tohoku University Graduate School of Medicine, Sendai, Japan, 2National Center of Neurology and Psychiatry, Tokyo, Japan
Email address for correspondence: [email protected]
Keywords: SOD1, CSPG, NG2
Background: Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Nerve/glial antigen 2 (NG2) is one of the major chondroitin sulfate proteoglycans (CSPG) upregulated after a variety of acute insults in the adult central nervous system (CNS), where they inhibit axonal regeneration and conduction.
Objectives: In order to clarify the role of NG2 proteoglycan under a chronic neurodegeneration such as ALS, we examined a temporal expression of NG2 proteoglycan and the NG2-expressing cell phenotypes in the spinal cord of a transgenic rat model of ALS.
Methods: The expression of NG2 proteoglycan was examined in spinal cord of His46Arg and Gly93Ala mutant SOD1 transgenic (Tg) rats at pre-symptomatic, early symptomatic, and late symptomatic stages with their age-matched non-transgenic (non-Tg) littermates. Continuous administration of a thymidine analogue bromodeoxyuridine (BrdU) for 7 days labelled newborn cells in vivo. After the administration, we performed multiple immunohistochemistry employing anti-NG2 specific antibody and cell-selective markers in the lumbar spinal cord cryosections. The immunofluorescence was digitally captured under confocal laser-scanning microscopy to determine the NG2-expressing cellular phenotype. In addition, we quantified the core protein expression levels by immunoblotting.
Results: In contrast to non-Tg rats, the 2 lines of Tg rats showed a significant and progressive accumulation of NG2 proteoglycan at the site of neurodegeneration in the ventral spinal cord. The multiple immunohistochemistry with cell-selective markers revealed that activated microglial cells, microvascular endothelial cells, and oligodendrocyte progenitor cells were suggested to constitute the NG2-expressing cells. In the gray matter, especially in the ventral horn, microglial cells were the major components of NG2-espressing cells and they often aggregated with the phagocytic features.
Discussion and conclusions: In the present study, we revealed a progressive accumulation of NG2 proteoglycan at the site of motor neuron degeneration in the rat ALS model. Recent reports have shown that the NG2-expressing microglial cells are involved in promoting neuroinflammation, and both the NG2 itself and inflammation can counteract the regenerative process in the damaged CNS. Therefore, NG2-expressing cells such as activated microglial cells may be considered as a potential therapeutic target in ALS.
P115 PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE DEFICIENCY DELAYS TONGUE MOTOR FUNCTION DEFICIT AND PROLONGS SURVIVAL IN THE SOD1-G93A MOUSE MODEL OF ALS
RINGER C, WEIHE E, SCHÜTZ B
Institute of Anatomy and Cell Biology, Marburg, Germany
Email address for correspondence: [email protected]
Keywords: PACAP, neuroprotection, microglia
Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleitropic functions, and expressed by many neurons throughout the central nervous system, including motoneurons. It has been reported that PACAP has neuroprotective properties, e.g. in preventing ischemic delayed neuronal cell death, and that it is a general anti-inflammatory factor, both in innate and adaptive immunity. Recently, it was shown that PACAP is able to protect rat motoneurons against glutamate-induced excitotoxicity in vitro (1), a pathomechanism discussed for ALS. Moreover, PAC1, the specific PACAP receptor, is expressed by astrocytes throughout the central nervous system, suggesting that astrocytes could also be a target of PACAP action in the process of non cell-autonomous motoneuron degeneration.
Objectives: We asked if a genetically induced PACAP deficiency (PACAP knockout) affects disease onset, progression, and motor functions in SOD1-G93A mice, the most frequently used mouse model of ALS.
Methods: We crossbred a PACAP-deficient mouse strain into the SOD1-G93A mouse model. Starting at postnatal day (P) 49, all mice (SOD1:PACAP−/−; SOD1/PACAP +/+; wt:PACAP−/−; and wt:PACAP + / +, 15-20 animals per group) were clinically monitored on a weekly basis, including measurement of body weight, paw grip endurance (PaGE) and licking motor tests, and finally survival.
Results: SOD1:PACAP−/− mice showed prolonged survival (142 +/− 12 days) compared to SOD1:PACAP +/+(131 +/− 7 days, p = 0,002). While both groups showed no differences in body weight loss and PaGE performance, tongue motor deficits, determined by licking frequency, were delayed in SOD1:PACAP−/− mice (mean onset at P112) compared to SOD1:PACAP +/+(mean onset at P105; p = 0.022). In addition, licking performance of SOD1:PACAP−/− also stayed significantly better than in SOD1:PACAP +/+ until end-stage.
Discussion and conclusions: Since PACAP deficiency prolonged survival of SOD1-G93A mice that was accompanied by a partially better motor performance compared to PACAP competent littermates, we conclude that PACAP may not generally be neuroprotective in vivo, at least in this ALS model. Differences to in vitro and in vivo observations made by others may be explained with additional deleterious effects of PACAP on innate and/or adaptive immunity, both contributing to disease. Since these effects could exceed neuroprotective autocrine pathways and be more important for ALS pathogenesis, they are currently under investigation.
Reference
- Tomimatsu N, Arakawa Y. J Neurochem 2008;107:628–635.
P116 SITE-SPECIFIC EXCITOTOXIN EXPOSURE IN VIVO LEADS TO NEURONAL EXCITOTOXICITY AND AXONAL DYSFUNCTION
BLIZZARD C, KING A, HOSIE K
Menzies Research Institute, Hobart, Tasmania, Australia
Email address for correspondence: [email protected]
Keywords: excitotoxicity, axonal degeneration, axonal dysfunction
Background: Despite recent advances, there still remain significant gaps in our current understanding of the mechanisms underlying axon degeneration in amyotrophic lateral sclerosis (ALS). ALS is likely to be a multifactorial disease of neuronal dysfunction and loss, however, recent investigations indicate that axonal dysfunction, prior to cell loss, may be the causative factor of the initial symptoms of ALS. Furthermore, it has been demonstrated in mouse models of familial ALS, other motor neuron diseases and, more recently, in studies of early human ALS, that distal axonal degeneration may occur before the onset of disease symptoms.
Objectives: Our investigations are focused on determining the degenerative changes underlying ALS-like axonopathy by using site-specific excitotoxic insults in vivo. We have developed a site-specific mouse model of excitotoxicity utilising osmotic minipumps (Alzet, model 1004), which will enable us to investigate the primary site of excitotoxic damage related to axonal pathology and ALS-like functional decline in vivo.
Methods: A constant and chronic infusion of Kanic acid (1-5mM, in cortex buffer) was delivered to the subarachnoid space of the lumbar region (L4-5) of C57/Bl6 mice and transgenic mice which express yellow fluorescent protein in a subset of motor neurons on a C57/BL6 background. Time and age matched controls were administered a constant infusion of cortex buffer. Fluro Ruby (2μM) was infused to determine the distribution of the Kanic acid. Animals were kept to a maximum of four weeks and all mice were terminally anaesthetised, transcardially perfused with 4% paraformaldehyde and then processes for immunohistochemistry to determine pathological changes occurring at the NMJ (distal axon), sciatic nerve (axon) and spinal cord (cell body and proximal axon).
Results: Fluoro Ruby labelling was present throughout cells within the subarachnoid space in L4-5 and a small number of neurons within the ventral horn, indicating a targeted delivery can be achieved with the osmotic pumps.
Discussion and conclusions: The data obtained by these experiments will enable us to investigate the primary site of excitotoxic damage related to axonal pathology and ALS-like functional decline in vivo. Identifying the site of the initial effects of excitotoxicity will identify mechanisms of distal axon degeneration that may provide novel therapeutic targets directed at axon protection.
P117 PULSATILE GROWTH HORMONE SECRETION IN THE SOD1G93A MOUSE MODEL OF ALS RESEMBLES GROWTH HORMONE DEFICIENCY IN ALS PATIENTS
NGO S1, STEYN F2, BUCKLEY A2, LEONG J2, VELDHUIS J3, CHEN C2, MCCOMBE P1,4, BELLINGHAM M2
1The University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia, 2The University of Queensland, Brisbane, Queensland, Australia, 3The Mayo Clinic, Rochester, Minnesota, USA, 4Royal Brisbane and Women’s Hospital, Brisbane, Queensland, Australia
Email address for correspondence: [email protected]
Keywords: growth hormone, endocrinology, SOD1G93A mouse
Background: The endocrine control of energy homeostasis is disrupted early in Amyotrophic Lateral Sclerosis (ALS), suggesting that this might contribute to ALS pathogenesis. ALS patients have increased basal energy expenditure, lean body mass index and muscle mitochondria abnormalities (1-5). These factors are thought to contribute to muscle wasting in ALS. Growth hormone (GH) is an anabolic hormone that aids in the development and maintenance of healthy muscle mass. Therefore, GH deficiency in ALS may also promote muscle wasting and be a consequence of the persistent hypermetabolic state in ALS. The mechanistic contribution to and effects of the disrupted GH axis in the loss of muscle mass in ALS however remains unknown. To further investigate these mechanisms we must first confirm that disruptions in the GH axis observed in ALS patients (6) also occurs in animal models that are used to study ALS.
Objective: To characterize the pattern of GH secretion in a transgenic mouse carrying the human SOD1 mutation.
Methods: Male wild-type and SOD1G93A transgenic mice were studied at the end-stage of disease (150-180 days). To determine one-off measures of plasma GH, blood was collected by cardiac puncture between 1530hrs and 1700hrs. To assess pulsatile GH secretion in mice, tail-tip whole blood samples (4μl) were collected consecutively over a 6hr period at 10min intervals starting at 0630hrs. An in-house GH ELISA was used to determine GH concentration. Data were analyzed by deconvolution analysis for properties associated with the pulsatile pattern of GH secretion.
Results: Analysis of single blood samples show that SOD1G93A end-stage mice and wild-type age-matched controls expressed similar plasma GH (n ≥ 12, p = 0.9449, t-test). By contrast, analysis of pulsatile GH secretion in samples collected over a 6hr period confirms a significant reduction in the total amount of GH secreted over the sampling period (n = 8 wild-type, n = 7 SOD1G93A). Furthermore, we observed a disruption in the pulsatile pattern of GH secretion. This was characterized by an overall decrease in the peak amplitude, and a loss of regularity of GH pulses. These observations suggest a disruption in the regulation of GH secretion in SOD1G93A mice at the end-stage of disease.
Discussion and conclusions: We report the first definitive account of disrupted GH secretion in a transgenic mouse model of ALS. Disrupted GH secretion in the SOD1G93A mouse closely resembles that seen in ALS patients. Defining the time course, causes and consequences of changes in GH secretion may lead to a greater understanding of what drives the onset and rate of progression of ALS symptoms, and can potentially provide new biomarkers and therapeutic strategies for ALS.
References
- Desport JC, et al. Am J Clin Nutr, 2001;74(3):328–34.
- Desport JC, et al. Neurodegener Dis, 2005;2(3–4):202–7.
- Dupuis L, et al. Lancet Neurol, 2011;10(1):75–82.
- Funalot B, et al. Amyotroph Lateral Scler, 2009;10(2): 113–7.
- Pellecchia MT, et al. Eur J Neurol, 2010;17(5):666–71.
- Morselli LL, et al. Clin Endocrinol, 2006;65(3):385–8.
P118 SYNERGY OF ERYTHROPOIETIN AND INSULIN-LIKE GROWTH FACTOR-1 CAN DETERIORATE CU,ZN-SUPEROXIDE DISMUTASE MUTANT AMYTORPHIC LATERAL SCLEROSIS VIA ACTIVATED MAMMALIAN TARGET OF RAPAMYCIN-INDUCED AUTOPHAGY INHIBITION
PARK SY1, SUNG J-J1, WANG R2, KANG Y-J2, LEE J-S1, KIM J-S1, YUN B-N1, MUN J-H1, KIM B-J1, KIM S-M1, HONG Y-H1, PARK K-S1, ZHANG D2, LIPTON SA2, LEE K-W1
1Seoul National University College of Medicine, Seoul, Republic of Korea, 2Sanford/Burnham Center for Neuroscience, California, USA
Email address for correspondence: [email protected]
Keywords: erythropoietin, insulin-like growth factor-1, autophagy inhibition
Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease presenting progressive motor weakness and respiratory failure within a few years from symptom onset. Many theories about pathogenesis are proposed including misfolded protein aggregation, mitochondrial dysfunction and oxidative cytotoxicity but exact pathogenic mechanisms are still not identified. Erythropoietin (EPO) and insulin-like growth factor I (IGF-1), show neuroprotective effects individually and have demonstrated to have synergistic neuroprotective properties in Alzheimer's dementia and Parkinson's disease when delivered simultaneously by activating phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) pathways. The possible limitation of their high-dose therapy causing systemic side-effect can be averted by detouring blood brain barrier (BBB) via delivering them through olfactory epithelium deficient of BBB. Autophagy is linked to neurodegenerative diseases as one mechanism for removing misfolded proteins. Target of rapamycin (mTOR) pathway, critical negative regulator of autophagy is located at downstream of PI3K/Akt pathway, of which activation could affect autophagy. However, their relationship to autophagy has not yet been elucidated. In this study, we evaluated the synergistic effect of EPO and IGF-1 simultaneously delivered in ALS using G93A Cu, Zn-superoxide dismutase (SOD1) mutant transgenic mice.
Method:We administered EPO and IGF-1 simultaneously to G93A SOD1 transgenic mice by transnasal delivery. Rotarod performance and survival analysis were assessed. Western blot and real-time PCR were done in order to investigate the drug mechanism. We then evaluated the effect of IGF-1 and EPO on the light chain 3 (LC3) dots representing autophagosomes by adopting GFP-LC3 transfected stable cell-line. We established NSC-34 cell line stably expressing human G93A SOD1 mutant, and assayed cytotoxicity.
Results: We found that EPO and IGF-treated G93A transgenic mice showed significantly poor performance on the rotarod task and earlier death compared with the mice treated with placebo. Western blot using antibodies against LC3, phosphorylated mTOR, and substrates of mTOR showed reduction of ratio of LC3-II /LC-I, and the activation of mTOR pathway. In-vivo imaging of GFP-LC3 expressing cells, rapamycin-induced LC3 dots were blocked by simultaneous treatment of IGF-1 and EPO. At the cell death assay, simultaneous treatment of IGF-1 and EPO induces more cell death in thapsigargin-induced cell death.
Conclusion: These findings showed that EPO and IGF-1 accelerate disease progression in G93A SOD1 transgenic mice compared with the placebo-treated group. The simultaneous treatment of EPO and IGF-1 activates mTOR pathway resulted in the decrease of autophagy. It could be a possible explanation of the deterioration caused by EPO and IGF-1, since autophagy is thought to remove mutant SOD1 aggregation. This EPO and IGF-1 mediated autophagy inhibition means that mTOR can be activated by the synergistic activation of PI3K/Akt pathway. It implicates that the inter-relationship between PI3K/Akt and mTOR pathways should be considered thoughtfully in order to develop new drug to target these pathways.
P119 DYSREGULATION OF INTRACELLULAR COPPER HOMEOSTASIS IS COMMON FOR DISMUTASE ACTIVE AND INACTIVE FORM OF SOD1 MUTANTS
TOKUDA E1, OKAWA E2, WATANABE S2, ONO S-I2,3, MARKLUND SL1
1Department of Medical Biosciences, Umeå University, Umeå, Sweden, 2School of Pharmacy, Nihon University, Chiba, Japan, 3Division of Neurology, Akiru Municipal Medical Center, Tokyo, Japan
Email address for correspondence: [email protected]
Keywords: intracellular copper homeostasis, TTM, dismutase active and inactive SOD1 mutants
Introduction: Mutations in the SOD1 gene cause familial ALS through a gain of toxic property. We have recently shown that dysregulation of intracellular copper homeostasis contributed to disease progression in a mouse model of ALS, which overexpresses the dismutase active G93A mutant SOD1 (1). However, it is unknown whether dysregulation of intracellular copper homeostasis is common for both dismutase active and inactive SOD1 mutants.
Objectives: The aim of the present study was to elucidate whether dysregulation of intracellular copper homeostasis could be a common feature in mutant SOD1 toxicity.
Methods: We used four different transgenic mouse strains overexpressing human SOD1 with the ALS-causing mutations: SOD1G93A, SOD1D90A, SOD1G85R and SOD1G127insTGGG (SOD1G127X). For each mutant SOD1 mouse model, spinal cords and brain were harvested from three end-stage mice of each genotype. C57BL/6J mice were used as controls.
The copper levels in the spinal cords were measured using inductively coupled plasma mass spectrometry. Because intracellular copper homeostasis is tightly regulated by intracellular copper trafficking-related proteins, we analyzed the expression level of the proteins using Western blot.
For treatment with an intracellular copper chelator, ammonium tetrathiomolybdate (TTM), SOD1G93A mice were randomly assigned to receive a daily intraperitoneal administration with TTM (5 mg/kg) or phosphate buffered saline. Treatment was started at 13 weeks of age, after the SOD1G93A mice began to exhibit ALS-like symptoms.
Results: Incorporation of copper ion into the copper-binding site is essential for SOD1 dismutase activity. Among mutant SOD1 mice that we used here, the SOD1G93A and SOD1D90A retain dismutase activity, whereas the SOD1G85R and SOD1G127X lack the activity due to insufficient copper ligation and are also present at very low levels in the CNS (2). We found that spinal copper levels were significantly increased in dismutase active form of mutant SOD1s mice. Interestingly, elevated copper levels were also observed in the inactive forms even though copper ions are not bound to SOD1G85R and SOD1G127X.
Western blot analysis showed that all mutant SOD1s shifted intracellular copper homeostasis toward copper accumulation: Ctr1, a copper uptake transporter, was significantly increased, whereas Atp7a, a copper efflux pump, was decreased. No changes were found in the brains.
Treatment with TTM, starting at a symptomatic stage, significantly prolonged the mean survival of SOD1G93A mice by 11%. Similarly, treatment with TTM dramatically slowed disease progression by 40%. TTM restored the elevated spinal copper ions to a normal level in SOD1G93A mice.
Conclusions: Enzymatically active and inactive SOD1 mutants share dysregulation of intracellular copper homeostasis. TTM might be an attractive candidate drug for therapy to ALS patients with SOD1 mutations.
References
- Tokuda E, Okawa E, Ono S. J Neurochem 2009;111:181–191.
- Jonsson PA, Graffmo KS, Andersen PM, et al. Brain 2006; 129:451–464.
P120 TDP-43 PATHOLOGY IN MICE EXPRESSING A VERY LOW COPY NUMBER OF THE MUTANT HUMAN G93A SOD1 GENE WITH A VERY SLOW DISEASE PROGRESSION
DEITCH J, YANG S, HEIMAN-PATTERSON T
Drexel University College of Medicine, Philadelphia, PA, USA
Email address for correspondence: [email protected]
Keywords: TDP43, SOD1, copy number
Background: Transgenic (Tg) 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 pathology is evident as early as 30d old and degeneration proceeds rapidly. This time course makes it difficult to discern disease onset, and may not reflect events in human MN degeneration. We have developed Tg mice (VLE mice) expressing a very low number (4-5) of copies of the G93ASOD1 transgene. These mice do not show clinical signs of MN disease until 650d old, if at all, providing the opportunity to more clearly discern both the sequence and nature of the pathologic changes in the mouse model and compare it to human ALS.
Objective: To determine whether pathological changes in TDP-43 and related molecules characteristic of human ALS, which have not been detectible in the rapidly progressing mouse model, are present in very slowly progressing mice with the SOD1 mutation.
Methods: VLE mice were compared to typical G93ASOD1 Tg mice (HE mice) expressing a high number of copies (24) of the transgene. All are on the B6SJL/F1 background. The number of gene copies was confirmed by qPCR. Male and female VLE mice at approximately 250, 500 and 750d old and HE mice at 90 and 120 days old were examined along with non-transgenic littermates. Paraformaldehyde-perfused spinal cords were embedded in paraffin, sectioned, and immunostained with an antibody to TDP-43 (Proteintech, 1:500). Staining patterns in the MNs in the ventral horns of lumbosacral segments were determined. MNs were easily identifiable by their position and large (>30 μm) cell bodies.
Results: TDP-43 labeling in young (90 and 120 day old) HE mice was largely confined to the nucleus, as previously observed. Labeling was similar in 250 day old VLE mice, an age at which motor neuron loss is not yet significant, although some cytoplasmic labeling was beginning to become evident. In older VLE mice, TDP-43 is distinctly distributed in cytoplasmic accumulations.
Discussion and conclusions: Redistribution of TDP-43 from the nucleus into cytoplasmic accumulations is observed in human ALS spinal cord MNs, in both FALS and SALS cases, but has not been established in the SOD1 mouse model. We hypothesized that the rapid degeneration of MNs in these mice may obscure these cellular changes characteristic of human ALS. Examination of TDP-43 distribution in MNs of VLE mice, in which degeneration proceeds at a very slow rate, revealed cellular changes more in line with those observed in human ALS. This slowly-progressing model of MN degeneration may be more relevant to cellular pathological changes in human ALS.
P121 DEVELOPMENTAL ROLE OF RNA PROCESSING PROTEINS IN NEURODEGENERATION
PERES J, BERGERHOFF K, HOUART C
King's College London, London, UK
Email address for correspondence: [email protected]
Keywords: zebrafish, RNA processing proteins, in vivo imaging
Background: The most recent advances in understanding ALS pathology come from the study of the RNA processing proteins TDP-43 and FUS. These genes were found to be mutated in both familiar and sporadic ALS and the proteins found in aggregates associated with the disease, but so far their mode of action remains unknown.
Objectives: Our work intends to unravel the function of TDP-43 and FUS by establishing the zebrafish as a model for neurodegeneration mediated by these RNA processing proteins.
Methods: We are performing gain and loss of function studies of zebrafish homologues of TDP-43 (tardbp and tardbpl) and FUS by micro-injecting either RNA or DNA (gain), or antisense morpholinos (MO, loss). These manipulations allow us to address the developmental role of these proteins. We are also overexpressing the human forms of the wildtype (wt) genes and several different mutations found in ALS patients. Loss-of-function (LoF) of the zebrafish proteins combined with the gain-of-function (GoF) of nuclear and cytoplasmic human forms reveals their relative contribution to the toxicity of these proteins.
Using fluorescent tag proteins we are also performing live imaging to analyze the intracellular protein dynamics in real time.
Results: Embryos lacking tardbp and tardbpl function consistently showed pericardial oedema, poor circulation, slow mobility, and reaction to touch from 3 days post fertilization (dpf) onwards. Motor neuron axons are shorter, with abnormal branching.
In the fus LoF, the embryos also have pericardial oedema, hydrocephaly and bent tail. Embryos at 3dpf still react to touch and are able to swim, having mild locomotion problems compared to wt. The motor neuron axons are unaffected.
Triple knockdown (tardbp + tardbpl + fus) show a more severe phenotype than the single or double combinations. The embryos are immobile and do not react to touch. The motor neuron axons show abnormal branching and migratory problems; ectopic cell bodies are visible adjacent to the spinal cord.
The GoF of tardbp and fus both have similar phenotypes; shorter and bent trunk, small eyes and head, cell death. The locomotion and touch response are not affected.
Rescue of LoF phenotypes by overexpressing different human constructs for TDP-43 and FUS are underway.
Discussion and conclusion: The zebrafish is becoming a powerful model for studying the function of RNA processing proteins in neurodegeneration. The LoF studies show a phenotype in the locomotion in response to touch. The triple knockdown suggests a functional interaction between tardbp/tardbpl and fus. We are now currently analyzing the factors behind this phenotype, looking at the activity, stability and connectivity of the motor neurons and performing RNA profiling studies.
The GoF studies are encouraging and will help elucidate the function of these proteins during early development. The information gained in these studies will contribute to the understanding of ALS pathology and ultimately deliver potential therapeutic targets.
P122 LIVE IMAGING OF MOTOR AXONS IN ZEBRAFISH EMBRYOS EXPRESSING AN ALS-RELATED MUTATION OF HUMAN TARDBP
BRUSTEIN E, LISSOUBA A, KABASHI E, DRAPEAU P
Department of Pathology and Cell Biology, Université de Montréal, Montreal, QC, Canada
Email address for correspondence: [email protected]
Keywords: in-vivo imaging, motor axon, transgenic zebrafish
Background: Amyotrophic lateral sclerosis (ALS) is associated with loss of motor neurons, however the origin of the neural dysfunction remains unknown. Previous studies suggest that deficits in axon function precede motor neuron death.
Objectives: To study axonal morphology in the context of ALS we followed the motor axon arbor organization and growth, in-vivo, in zebrafish embryos expressing GFP under the motor neuron promoter HB9 and expressing the sporadic and familial ALS-related mutation G348C of human TARDBP under the inducible heat-shock Hsp70 promoter.
Methods: Trangenic zebrafish embryos were subjected to heat-shock at 24h post fertilization at 38.5°C for 30 minutes (sufficient to induce ubiquitous expression) and imaged 2-4 hours later, every 2 minutes for a total of 30 minutes using a spinning disk confocal microscope. The stacked images of the motor axons were then 3D reconstructed for each time point using Imaris software (Bitplane, USA). The 3D reconstruction provided information about main axon and secondary neurite length, density and motility. The results from transgenic embryos were compared to data obtained from control embryos including non-transgenic siblings that were heat- shocked and transgenic and non transgenic siblings that were not heat-shocked.
Results: Transgenic embryos imaged as soon as 2h hours after the heat-shock had hyper branched primary axons compared to control embryos which had one main axon. These results confirm data obtained previously (1) from embryos injected with the human mRNA carrying G348C mutation and fixed 48h post fertilization. In addition, the number of secondary neurites was reduced in the mutant, but their length was similar to that of the controls. The terminal endings of the main branches in control embryos were quite stable but were more motile in mutants, suggesting unstable innervation by supernumerary mutant axon branches.
Discussion and conclusions: Our study is first to follow axon structure and dynamics in-vivo in the context of ALS and our preliminary results suggest that the motor neuron axon deficits occurs soon after the expression of the mutated TARDBP protein, before motor neuron death.
References
- Kabashi E, Lin L, Tradewell ML, et al. Hum Mol Genet. 2010;19(4):671–83.
P123 DEFECTIVE NEUROMUSCULAR TRANSMISSION IN ZEBRAFISH EXPRESSING HUMAN TARDBP (TDP-43) WITH A MUTATION RELATED TO ALS
ARMSTRONG G, LIAO M, DRAPEAU P
Université de Montréal, Montreal, Quebec, Canada
Email address for correspondence: [email protected]
Keywords: zebrafish, TDP-43, electrophysiology
Background: Mutations in the TARDBP gene encoding TDP-43 have been found in ALS and FTLD patients. Although several animal models for these TDP-43 mutations have been described, none have characterized the pathophysiological deficits that underlie the resulting phenotype.
Objectives: To advance our understanding of the neurophysiological deficits resulting from mutations in the TARDBP gene encoding TDP-43.
Methods: We used a zebrafish model previously described by our lab (1) in which we transiently expressed mutant human TARDBPG348C as well as wildtype TARDBPWT mRNA in zebrafish larvae. Standard Immunohistochemistry and patch-clamp recordings were performed on muscle and motor neurons in larvae aged 48 hrs.
Results: Following over expression of mutant but not WT TDP-43 we observed specific deficits at the neuromuscular junction (NMJ). Immunohistochemistry revealed hyperbranched motor neuron endings with AChR clusters at NMJs in fish expressing TARDBPG348C but not TARDBPWT or WT fish. We next examined spontaneous miniature endplate currents (mEPCs) related to the release of single transmitter quanta at the NMJ. Two striking features were observed: the amplitude of mEPCs was significantly larger and occurred at a lower frequency in fish expressing mutant TARDBPG348C but not WT TARDBP. We next evoked swimming-related activity and observed normal rhythmic EPCs at NMJs from fish expressing TARDBPWT. In contrast, fish expressing TARDBPG348C displayed a slower frequency of rhythmic EPCs with shorter bouts of swimming-related activity. In paired recordings action potentials were elicited in motor neurons while recording EPCs in the muscle. We observed an impairment in the fidelity of sustained synaptic transmission.
Discussion and conclusions: These data represent the first electrophysiological description of TARBP-related mutations and indicate that over branched motor neurons form NMJs each with exaggerated spontaneous release and yet overall weaker sustained release at the NMJ.
Reference
- Kabashi E, Lin L, Tradewell ML, et al. Hum Mol Genet 2010;15:671–683.
P124 TDP-43 TRANSGENIC C.ELEGANS DEVELOPED MOTOR DYSFUNCTION CHARACTERISTIC OF ALS
LI W, LI Z, WU Q, YE C, LU X, YAO X
Department of Neurology, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
Email address for correspondence: [email protected]
Keywords: TDP-43, C.elegans
Objective: To deliver the expression plasmid:unc-51/hTDP-43 by microinjection to the gonad of C.elegans to establish a TDP-43 ALS model and to investigate the mechanism of TDP-43 motor neurotoxicity, including dissection of which molecular features of TDP-43 pathology cause motor neuron degeneration.
Methods: To generate transgenes expressing human TDP-43 driven by a pan-neuronal unc-51 promoter, which were Punc-51::TDP-43(WT) Punc-51::TDP-43(G348C), Punc-51::TDP-43(S292A), and Punc-51::TDP-43 (CTFs) respectively. The transgenes described above were microinjected into hermaphrodite gonads of N2 worm at a concentration of 50-100μg/ul to generate multiple extrachomosomal lines based on the green fluorescent marker. The locomotion of control and transgenic worms was assayed by picking 10-20 L4 or adult worms onto 100 mm nematode growth medium (NGM) agar plates spread with a uniform bacterial lawn. Worm locations were then recorded at the indicated time points, and linear distances from the starting position were measured. And the neurodegeneration assays were performed that timed egg lays were arranged to produce synchronized populations 8, 24, and 48 h of age. Live worms were placed on a 3% agarose pad containing 0.01% sodium azide to immobilize the worms. Worms were imaged under fluorescence microscopy and scored for number of dorsal motor neurons, and gaps in the dorsal nerve cord. Finally, this ALS model was examined by Western blot. In order to analyze the cellular localization of TDP-43, immunocytochemistry was performed.
Results: By gene clone, we successfully constructed hTDP- 43 expressive vectors, they are pU51P::hTDP-43(WT), pU51P::hTDP-43(G348C),pU51P::hTDP-43(S292A), pU51P::hTDP-43(CTFs) respectively. By microinjection and offspring screening, we obtained the stable inherited ALS model of TDP-43 proteinopathy. The biological characteristics of this ALS model: typical motor dysfunction; the degeneration and loss of motorneuron synapses; and TDP-43 proteins aggregated in the nucleus of neurons.
Conclusions: Use of C.elegans as a model system permits unbiased approaches to genetically manipulate a model of human neurodegenerative diseases. This TDP-43 ALS model is likewise tractable for genome-wide forward and reversed genetic screens, which will allow identification of novel modifiers of TDP-43.
P125 SPATIAL CHARACTERIZATION OF THE MOTOR NEURON COLUMNS SUPPLYING THE RAT FORELIMB
TOSOLINI A, MORRIS R
University of New South Wales, Sydney, NSW, Australia
Email address for correspondence: [email protected]
Keywords: rat models, motor neurons, cervical spinal cord
Background: Rats can generate a rich array of forepaw and forelimb movements that are similar to those produced by humans, therefore making them attractive models to validate therapeutic intervention for the recovery of motor control. We are currently exploring strategies using virus to deliver therapeutic genes to specific populations of motor neurons in the rat. This can be achieved via intramuscular injections of viral vectors and subsequent retrograde transport and expression of the therapeutic gene in targeted motor neuron populations. In order to achieve this, knowledge regarding the precise relationship between different muscles of the forelimb and the location of motor neurons that innervate them must be first established.
Objectives: This study aims to examine the details of the arrangement of motor neurons that supply the rat forelimb.
Method: Eleven upper limb muscles from the rat were selected. The muscle motor end plates were visualized by means of acetylcholinesterase histochemistry and this information was then used to create a motor end plate map of the forelimb. This map was used as a guide to perform multiple injections of fluorescent retrograde tracers (Fluoro-Gold and Fluoro-emerald) along the motor end plate region of the selected forelimb muscles. 12-14 days later, the rats were perfused intra-cardially and the spinal cord segments of interest were dissected out, sectioned and analysed under epifluorescence. For each muscle, the positively labelled motor neurons were plotted on a schematic reconstruction of the spinal cord. The individual plots were then stacked in order to create motor neuron maps in all axes.
Results: This tract-tracing analysis confirmed that motor neurons innervating the rat forelimb are arranged in columns that span across multiple spinal cord segments. Individual motor columns exhibit a substantial degree of overlap with other motor columns in all planes (i.e. rostro-caudal, dorso-ventral and medio-lateral axes).
Discussion and conclusions: This anatomical investigation supports previous observations that, although discrete, some of the motor neuron columns lying in the cervical aspect of the rat spinal cord are inter-digitized. The length of these columns, and hence the overlap between them, appears to be greater than previously reported, particularly within the uppermost segments of the brachial plexus. This map constitutes a valuable guide for the selection of appropriate muscle(s) for the delivery of therapeutic genes into specific segments of the cervical spinal cord. The organization of the motor neurons supplying the rat forelimb may have significant applications for the development of therapies in rat models of forelimb dysfunctions including models of motor neuron diseases.
P126 A REDUCED REQUIREMENT FOR SMN, THE SPINAL MUSCULAR ATROPHY PROTEIN, DURING ADULT LIFE
KARIYA S, IWATA S, HOMMA S, MONANI U
Columbia University, New York, NY, USA
Email address for correspondence: [email protected]
Keywords: spinal muscular atrophy, SMN protein, therapeutic window
Prior observations of the effects of reduced Survival Motor Neuron (SMN), the underlying cause of the childhood disorder, Spinal Muscular Atrophy (SMA), have been made largely in embryos and neonates. The requirement for the protein during adult life has not been investigated. We and others have shown that reduced protein arrests the development of one component of the neuromuscular system - the neuromuscular synapse, raising the possibility that once the synapse is mature, depleting protein, e.g., in adults may not adversely affect the organism. To test this possibility, we have generated model mice harboring a tamoxifen-responsive Smn allele (SMNF7) and human SMN2 transgene and ubiquitously reduced the SMN protein at different time points during postnatal life.
Our results demonstrate, for the first time, that the requirement for SMN is higher in young mice than in adults. Surprisingly, depleting protein in adults carrying 2 copies of the human SMN2 (Cre-ER +/−;SMNF7/−;SMN2 +/+) to severe SMA levels appeared not to result in an overt phenotype whereas a similar reduction in young animals caused progressive muscle weakness.
These data suggest that there is a window of time in postnatal life during which wild-type levels of the SMN protein are required for the normal development of the neuromuscular and/or other systems, following which cellular demand for the protein drops. An important implication is that infants diagnosed with SMA in a timely manner may not require chronic treatment with SMN restoring agents and thus the prospect of possible adverse, drug-related side-effects. Rather, a limited treatment regimen during a critical window of time could be sufficient to allow the proper development of the neuromuscular system and prevent an SMA phenotype from eventually developing. Further defining the window of time for SMN requirement and correlating it with the development of various organ systems will inform our understanding of the precise cellular site(s) of action of the protein in health and disease and thus contribute to effective future treatments for SMA patients.