Society for Neuroscience Abstracts
	1999 IDENTIFICATION OF A NOVEL EXCITOTOXIN FROM CYCAD SEED IMPLICATIONS FOR NEURONAL DISORDERS. C.A Shaw'.. JS. Bains'. D.E Williams'. RJ Andersen'. BA Pasaualotto'. J Cheune1. M. Tiandrawidiaia'. M Wilkinson'. R. Janakv'. and U-K Craig Depts. of 'Ophthalmology, 'Chemistry and Earth and Ocean Sciences, and 'Physiology, Univ. British Columbia, Vancouver, B.C.; Dept. 'ObiGyn, Dalhousie Univ., Halifax, N.S;"fampere BmiR Research Center. Tampere Univ. Medical School, Tampere, Finland; and Lytico and Bodig Research Project', Univ of Guam One causal factor in various neurological disorders may be exposure to particular neurotoxins, specifically those which may over excite neurons leading to excitotoxic cell death We have examined this hypothesis in regard to the potential toxicity of flour made from cycad seed in relation to a neurological disorder endemic to Guam; ALS-parkinson's dementia complex (ALS-PDC) Flour from cycad (Cycas circinalis) was prepared by traditioual methods and a methanol extract prepared 'Crude', aqueous, or ethyl acetate (organic) sub- fractions were isolated Fractious were assayed for functional activity by electrophysiological recording in an in vitro rat cortical slice preparation, by receptor binding in crude synaptic membrane homogenates, and by calcium influx in cerebellar granule cells in culture Toxicity was assessed by LDH release and apoptosis (TUNEL) in rat cortex slices or heat shock protein (HSP-25) expression in young rat hypothalamus. Based on results of tile assays, active fractions were further separated by column chromatography and HPLC until a single compound could be isolated and examined by NMR The results reveal that washed cycad flour contains various excitotoxic fractions, some of which appear to act as agonists at the NMDA receptor 1LC and NMR show that these compounds are not those traditionally associated with cycad toxicity, ie, BMAA or cycasin/MAM Instead the most active of these appears to be an unusual steroid with a MW of approx 600 The present data suggest the possibility that this compound may be a factor leading to neural degeneration in ALS-PDC, a hypothesis that will be tested in an in vivo animal\ model These studies were supported by an ALS Association grant to CAS. SOCIETY FOR NEUROSCIENCE, VOLUME 25, 1999 2000 MECHANISMS OF ACTION OF SITOSTEROL GLUCOSIDE IN MAMMALIAN CNS. Khabazian^1; S.L. Pelech^2; D.E. Williams^3; R.J. Andersen^3; U.-K. Craig^4; C. Krieger^5*; C.A. Shaw^1 1. Department of Ophthalmology, 2. Department of Medicine, 3. Department of Oceanography, University of British Columbia (UBC), Vancouver, BC, Canada, 4. University of Guam, Mangilao, Guam, 5. Department of Kinesiology, Simon Fraser University, Burnaby, BC, Canada. The etiology of the cluster of neurological disorders on Guam, ALS-PDC, remains unknown. However, epidemiological evidence links the disorder to consumption of the seed of the cycad (Cycas circinalis) through the action of a putative neurotoxin. We have examined the toxicity of various cycad fractions separated by HPLC in an in vitro rat cortical slice preparation. The active component in cycad was -sitosterol--D-glucoside (BSSG), of which three closely related forms were found. Each of these fractions was found to induce activation through NMDA receptors leading to depolarizing field potentials. BSSG also increased PK activation, glutamate release, and increased cell death qualitatively similar to that produced by exposure to NMDA. Each of the action of BSSG was blocked by the NMDA antagonist AP5. We have also synthesized BSSG and compared its actions to the native BSSG and found these to be identical in action and toxicity. The identification of a potent neurotoxin in cycad identified as BSSG raises the possibility that this substance may play a key role in the etiology of ALS-PDC. Supported by: ALS Association (US) and Scottish Rite Charitable Foundation NECROTIC AND APOPTOTIC CELL DEATH DUE TO NEUROTOXIC ACTIONS OF MSO AND CYCAD. R.J. Simpson1; I. Khabazian1; J.S. Bains1; D.E. Williams2,3; R.J. Andersen2,3; U.-K. Craig4 ; C.A. Shaw1* 1. Ophthalmology, 2. Earth and Ocean Sciences, 3. Chemistry, University of British Columbia, Vancouver, BC, Canada 4. University of Guam, Agana, Guam To investigate the hypothesis that novel environmental neurotoxins contribute to the etiology of neurodegenerative diseases, we have compared the forms of cell death initiated by the following: (i)methionine sulfoximine (MSO); (ii) an extract of cycad flour; and, (iii) the putative neurotoxic component of cycad, -sitosterol -D-glucoside (BSSG). To measure acute necrosis, experimental compounds were applied to adult rat cortical slices in vitro and the amount of lactate dehydrogenase (LDH) release was measured as a direct indication of the levels of cell death. In a second series of experiments, apoptosis was assessed by immunocytochemical staining using an adapted TUNEL method on sections of frontal cortex, hippocampus, spinal cord and retina of rats fed MSO (3, 6, 9 mg/rat/day) or cycad flour (2g/rat/day) for two weeks and compared with controls. Both MSO, cycad flour extract and BSSG induce rapid LDH release in cortical sections. Neither MSO nor cycad gave significant neuronal apoptosis, but the latter gave apoptosis in the pigment epithelium. Supported by: ALS Association (US), Scottish Rite Charitable Foundation, NSERC 2001 HISTOLOGICAL ASSESSMENT OF CYTOTOXICITY DUE TO CYCAD FEEDING IN MICE: IMPLICATIONS FOR ALS-PDC. J.M.B. Wilson^1; M.C. Wong^2,3; A. Seyedalikhani^1; B.A. Pasqualotto^4; C.A. Shaw^2,3,4* 1. Psychology, University of British Columbia, Vancouver, BC, Canada 2. Ophthalmology, University of British Columbia, Vancouver, BC, Canada 3. Neuroscience Programme, University of British Columbia, Vancouver, BC, Canada 4. Physiology, University of British Columbia, Vancouver, BC, Canada Consumption of cycad seed products (Cycas circinalis) has been implicated as a causative factor to the Guamian neurological disorder, ALS-PDC. Using in vitro rat cortical slice assays, we demonstrated that washed cycad contains a cytotoxic sterol glucoside; this molecule causes glutamate release leading to activation of NMDA receptors (Shaw et al., 1999). In vivo studies in mice have shown cognitive and motor behavioural deficits following consumption of cycad flour (Wilson et al., 2001). We performed a series of histological studies designed to link sterol glucoside in vitro toxicity to in vivo evidence of CNS dysfunction. Adult male mice were fed washed cycad flour then sacrificed following 7 weeks of behavioural testing. TUNEL, caspase-3, HSP-70 and tau assays were performed in search for evidence of neurodegeneration. Anti-tau immunolabeling (cortex and hippocampus) was 2x more abundant in cycad fed-mice. In cycad-fed mice, TUNEL labeled cells were found in cortex, hippocampus, spinal cord and substantia nigra (SN) and anti-HSP-70 antibody labeled cells were found in regions of CNS. Anti-activated Caspase-3 immunostaining was found in the SN of one cycad-fed animal. These findings support the view that the cytotoxic effects of cycad flour is the basis of the behavioral deficits observed in cycad-fed mice and the hypothesis that the etiology of Guamian ALS-PDC involves ingestion of a toxin in cycad flour. Supported by: ALSA and Scottish Rite Charitable Fdn of Canada 2002 EFFECTS OF CYCAD TOXINS ON GLUTAMATE TRANSPORTERS IN A MURINE MODEL OF ALS-PDC. I.Khabazian^1; D.V. Pow^2; C. Krieger^3*; C.A. Shaw^1 1.Ophthalmology, The University of British Columbia, Vancouver, BC, Canada 2. School of Biochemical Sciences, University of Queensland, Queensland, Australia 3. Kinesiology, Simon Fraser University, Burnaby, BC, Canada We have previously shown that toxins extracted from seeds of the cycad Cycas circinalis act as neurotoxins in an in vitro rat cortical slice preparation (1). To further explore the mechanism of cycad-induced toxicity, we fed washed cycad flour to mice while assessing both motor and cognitive functions (2). Behaviorally and neurologically, the outcomes resembled key features of ALS Parkinsonism dementia complex (ALS-PDC). Since abnormalities of glutamate transporters are a feature of Alzheimer's disease (3; 4) and ALS (5), Western blotting was employed to examine the levels of these proteins in the central nervous system of mice fed with cycad. A significant apparent decrease (-68%) was observed in a 30 kDa fragment of GLT-1B (a C-terminal splice variant of GLT-1) in cycad fed mice. No significant changes were detected in protein concentration levels for another splice variant of GLT-1 (GLT-1) (6). Further experiments were conducted to determine if the apparent change in GLT-1B protein levels observed corresponded to the hosphorylation state of this transporter. Alkaline phosphotase co-incubation with cortical homogenates gave equivalent levels of GLT-1B transporter in cycad-fed and control mice, suggesting that the altered state of in cycad-fed mice arises from abnormally phosphorylated GLT-1B. These data support the view that functional alterations in specific glutamate transporters by phosphorylation play a role in the neurodegenerative processes following exposure to cycad toxins. (Supported by: ALS Association (US), Scottish Rite Charitable Foundation). Citation: I. Khabazian, D.V. Pow, C. Krieger, C.A. Shaw. EFFECTS OF CYCAD TOXINS ON GLUTAMATE TRANSPORTERS IN A MURINE MODEL OF ALS-PDC. Program No. 44.1. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online. THE EFFECTS OF INTRACRANIAL NEURAL STEM CELL TRANSPLANTATION ON NEUROPATHOLOGICAL OUTCOMES INDUCED BY CYCAD TOXINS J. Lai^1; J.K. Ryu^2; V. Lukic^1; P.T.-L. Kuang^1; S.U. Kim^2; C.A. Shaw^1* 1.Ophthalmology, 2. Neurology, University of British Columbia, Vancouver, BC, Canada Much recent research has focused on the potential for therapy of stem cell injections directly applied to the site of CNS damage in neurodegenerative diseases. We have developed an environmental model of Amyotropic Lateral Sclerosis-Parkinsonian Dementia Complex (ALS-PDC) by feeding mice flour made from the seed of cycad, a plant found in Guam that contains a novel neurotoxin. Previous studies in our laboratory showed robust behavioral and neurological deficits in mice resulting from cycad ingestion. In the present study, experimental animals received daily supplements of cycad pellets (n=14) and control animals received commercial flour pellets (n=14) until animals showed significant behavioral deficits. We employed the Morris Water maze, leg extension reflex and a novel olfactory test as indices of spatial memory, motor neuron integrity and olfactory bulb function, respectively. Animals either received stereotaxic injections of human neural stem cell line F3 or PBS (both n=7 cycad and n=7 control) bilaterally into the lateral ventricles. Behavioral testing was continued for 6 weeks post-injection to allow time for neural stem cell migration, maturation and integration. Histological data showed that stem cells migrated to cortex, striatum, hippocampus and olfactory bulb. Furthermore, slices were stained for Nestin, NeuN, GFAP and MBP for morphological analysis. Confocal microscopy revealed differentiation of stem cells into neurons and glia. To assess the potential of stem cell therapy on behavior, performance on behavioral tasks were correlated with histological findings. Supported by: ALS Association, NSERC Citation: J. Lai, J.K. Ryu, V. Lukic, P.T.-L. Kuang, S.U. Kim, C.A. Shaw. THE EFFECTS OF INTRACRANIAL NEURAL STEM CELL TRANSPLANTATION ON NEUROPATHOLOGICAL OUTCOMES INDUCED BY CYCAD TOXINS Program No. 34.9. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online. SEX DIFFERENCES IN NEUROLOGICAL DEFICITS INDUCED BY CYCAD IN A MOUSE MODEL OF ALS-PDC M.C. Wong^1*; C.A. Shaw^1,2; J.M.B. Wilson^1; A. Seyedalikhani^2 1.Dept of Neurosci, 2. Ophthalmology, Univ of British Columbia, Vancouver, BC, Canada Patients on the island of Guam suffering from Amyotrophic Lateral Sclerosis - Parkinsonism Dementia Complex (ALS-PDC) may show symptoms of ALS, Parkinson's disease, Alzheimer's disease or any combination of the three. The incidence of this disease is highly correlated to the consumption of flour made from the seed of cycad (Cycas circinalis). Motor, cognitive and sensory deficits as well as CNS tissue abnormalities result from consumption of cycad flour pellets in a mouse model of ALS-PDC (Wilson et al., 2002). A putative water insoluble toxin in the cycad flour that may be responsible for these deleterious changes has been identified (Khabazian et al., 2002). To examine if the model also reflects the sex differences in severity and incidence of disease, cycad flour pellets were fed to intact adult male and female mice. Motor deficits were found be greater in male than female mice fed cycad, which may correlate with the higher incidence of ALS-PDC in human males. However, olfactory deficits were greater in females than males. The results of this study are important for validating the accuracy of the model as well as implicating gonadal hormones, especially estrogen, in ALS-PDC and other related neurological disorders. Supported by: ALSA and Scottish Rite Charitable Fdn. of Canada Citation: M.C. Wong, C.A. Shaw, J.M.B. Wilson, A. Seyedalikhani. SEX DIFFERENCES IN NEUROLOGICAL DEFICITS INDUCED BY CYCAD IN A MOUSE MODEL OF ALS-PDC Program No. 205.14. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online. 2003 A MURINE MODEL OF ALS-PDC: BEHAVIOURAL AND NEUROPATHOLOGICAL FEATURES OF PARKINSONISM. J.D.Schulz^1*; E.L.Hawkes^1; C.A.Shaw^1,2 1.Grad. Program in Neurosci., Univ. of British Columbia, Vancouver, BC, Canada 2.Ophthalmology, Univ of British Columbia, Vancouver, BC, Canada The consumption of flour made from the seed of Cycas micronesica, a species of cycad, has been linked to the unusual neurological disorder ALS-Parkinsonism dementia complex (ALS-PDC). In support of the hypothesis that cycad has a causal role in this disorder, adult mice fed cycad flour develop behavioral and histopathological outcomes that mimic ALS-PDC (Wilson et al., 2002). To examine whether cycad neurotoxicity is causal to the parkinsonism aspect of ALS-PDC, mice were fed washed cycad flour as part of their diet and behavioral and histological assays were used to assess parkinsonism-related pathology. Gait length, an indicator of basal ganglia function that correlates to cell loss in the substantia nigra and striatum, was used to monitor deficits in motor function. Striatal-dopaminergic integrity was assessed by immunohistochemical detection of tyrosine hydroxylase (TH) and apoptosis was detected by TUNNEL. Animals fed washed cycad flour showed a significant, progressive motor deficit in gait length that was on average 15% shorter than control animals. Assays for TH immunoreactivity revealed a significant 16% decrease in striatal dopaminergic terminals of the cycad-fed mice. In some animals there was also a decrease in substantia nigra TH immunoreactivity, although this did not attain statistical significance. In addition, apoptotic cells were observed in the substantia nigra of cycad- fed animals. Further histological results are pending, including assays for activated microglia, oxidative stress and -synuclein, which should provide further clues as to the nature of cycad-induced neurodegeneration. These results show that our murine model of ALS-PDC includes both behavioral and neuropathological features of parkinsonism. This further validates the use of this model in studies of ALS-PDC and Parkinsons disease. Support Contributed By: PDF, ALSA, NSERC, USAMRMC Citation: J.D. Schulz, E.L. Hawkes, C.A. Shaw. A MURINE MODEL OF ALS-PDC: BEHAVIOURAL AND NEUROPATHOLOGICAL FEATURES OF PARKINSONISM. Program No. 948.8. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. CHOLESTEROL -D-GLUCOSIDE: A NOVEL PUTATIVE CAUSAL FACTOR IN ALS-PDC. Khabazian^1*; J.Kyu Ryu^2; A.Nagai^3; S.U.Kim^2; C.A.Shaw^1 1. Dept. Opthalmol, Univ. British Columbia, Vancouver, BC, Canada 2. Dept Med., Univ British Columbia, Vancouver, BC, Canada 3. Dept Intrnl. Med., Shimane Med. Univ., Izumo, Japan ALS Parkinsonism dementia complex (ALS-PDC) is a disease that had been predominant in Guam for decades. We study this disease as a model that could enhance our understandings of other neurological diseases in elsewhere. The epidemiological studies link this disease to the environmental toxins in the seeds of Cycas circinalis (1). Extracted toxins from cycad flour on cortical slices in an in vitro preparation led to glutamate release and cell death (2, 3). Mice fed with cycad flour showed both cognitive and motor deficits resembling ALS-PDC (4). The extracted toxins were resolved by nuclear magnetic resonance (NMR) and mass spectrometry (MS) as sterol -D-glucosides (SSG); synthetic SSG and cholesterol |*beta*-D-glucoside (CG) were also largely neurotoxic (2). To better understand the possible mechanism of action of CG and its role in activating biochemical cascades that lead to glutamate release and cell death, we prepared a mixed culture of telencephalic neuronal and astroglial cells isolated from human fetal brain; these cells are interdependent in their growth and morphology. Fluorescent immunocytochemical labeling against cytosolic proteins Caspase-3, HSP-70, PHF-Tau, and GLT-1|*beta* were measured in relation to time and to each other. We also assessed activation patterns of these proteins based on treatment time and post incubation time. The activation of HSP-70 rises fast and decreases over time, while that of Caspase-3 is the reverse and stands in the opposite side of the spectrum. The expression of GLT-1|*beta* and PHF-Tau seem to take place somewhere in between activation time of HSP-70 and Caspase-3 relative to time. These data support the view that functional alterations in specific cytosolic proteins play a big role in the neurodegenerative processes following exposure to CG. Support Contributed By: US ALS and US Dept Defense Citation: I. Khabazian, J. Kyu Ryu, A. Nagai, S.U. Kim, C.A. Shaw. CHOLESTEROL -D-GLUCOSIDE: A NOVEL PUTATIVE CAUSAL FACTOR IN ALS-PDC. Program No. 630.14. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. CYCAD TOXICITY IN A FEMALE MOUSE MODEL OF ALS-PDC. M.C.Wong^1*; V.Lukic^2; C.A.Shaw^1,2,3 1. Neurosci., 2. Ophthalmology, 3. Physiology, Univ. of British Columbia, Vancouver, BC, Canada At the peak of its prevalence, Amyotrophic Lateral Sclerosis Parkinsonism Dementia Complex (ALS-PDC) was approximately twice as common in males as compared to females. ALS-PDC is a neurological disease that has previously been shown to be highly correlated to the consumption of cycad (Cycas micronesica) flour. To examine whether cycad causes toxicity in female mice as it does in male mice, we fed intact adult female CD-1 mice 0.5g of cycad flour daily for 28 days and tested them for various motor abilities. The mice were tested for 7 consecutive days at the following times: before cycad feeding, immediately after the 28 days of feeding, 4 weeks after the cessation of feeding and prior to sacrifice. Control mice were fed 0.5g of commercial white flour. We used leg extension to test for reflex, the paw print test for gait disturbances and wire hang for upper limb strength. Weights of the mice were also recorded. No weight differences were found between control and experimental animals, nor was there a difference within the groups across time. Cycad-fed animals performed considerably worse than control animals on the leg extension test by week 7. Mice fed cycad also had poorer performance on the wire hang test by week 13. Both effects remained stable throughout the remainder of the experiment. Differences were not observed for gait length, indicating that this aspect of motor ability is preserved in female mice fed cycad. Overall, cycad was found to induce motor deficits in female mice. However, unlike what is observed in male mice, cycad consumption did not induce a decrease in gait length in female mice. This finding suggests that some aspects of motor functioning may be protected from cycad toxicity in females. Support Contributed By: ALSA, SCRF, NSERC, USAMRMC Citation: M.C. Wong, V. Lukic, C.A. Shaw. CYCAD TOXICITY IN A FEMALE MOUSE MODEL OF ALS-PDC. Program No. 630.12. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. QUANTITATIVE MEASUREMENT OF NEURODEGENERATION IN ALS-PDC MODEL USING MRI. M.S.Petrik^1; J.M.B.Wilson^2; S.C.Grant^4; S.J.Blackband^4; C.A.Shaw^1,2,3* 1. Ophthalmology, 2. Neurosci., 3. Physiology, Univ. of British Columbia, Vancouver, BC, Canada 4. Neurosci., Univ. of Florida, Gainesville, FL, USA Cycad (Cycas micronesica K.D. Hill) feeding has been shown to induce neurodegeneration in vivo that mimics the progressive neurological disease, ALS-PDC (Wilson et al., 2002). Previously, specific cortical and subcortical cell loss was measured with stained 2D sections. In this study, MRI images were used to examine neurodegeneration in 3D. Mice were fed washed cycad as part of their normal diet for 2 months and showed progressive motor and cognitive behavioral deficits resembling human ALS-PDC. Animals were perfused and CNS tissue was scanned using a 750MHZ MRI with a 17.6T magnet. T2* and diffusion tensor scans were conducted on both spinal cord and brain samples. MRI data files were then analyzed using Amira 3.0 software to segment out areas of interest and measure volumes and cortical thickness. Resolution of the scans was ~40m. Cycad-fed mice showed significantly decreased substantia nigra, striatum, and olfactory bulb (granular layer) volumes. Cycad-fed mice that received stem cell injections displayed decreases in these same areas. Cortical measurements revealed that the cycad-fed mice also showed decreased cortical thickness in primary and secondary somatosensory cortexes, including hind limb cortex. Diffusion tensor imaging demonstrated changes in regions of the brains and spinal cords of cycad-fed mice, which may indicate an alteration in axonal tracts. These results show that MRI imaging is sensitive enough to measure degeneration in early stage model of a progressive neurological disease. The merits of this analysis may be used in the future as a diagnostic aid in tracking the progression of neurological disease, and serve as a tool for future neurological disease research. Support Contributed By: ALS Assocation, SRCF, NSERC, and the US Army Medical Research and Materiel Command (#DAMD17-02-1-0678) (to CAS) Citation: M.S. Petrik, J.M.B. Wilson, S.C. Grant, S.J. Blackband, C.A. Shaw. QUANTITATIVE MEASUREMENT OF NEURODEGENERATION IN ALS-PDC MODEL USING MRI. Program No. 630.10. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. RECOVERY OF MOTOR FUNCTION IN MICE FED CYCAD TOXINS: IMPLICATIONS FOR ALS? E.L.Hawkes^1*; J.D.Schulz^1; C.A.Shaw^1,2 1.Grad. Program in Neurosci., Univ. of British Columbia, Vancouver, BC, Canada 2.Ophthalmology, Univ. of British Columbia, Vancouver, BC, Canada Cycad toxins have been implicated in the Guamanian neurological disease, ALS-parkinsonism dementia complex (ALS-PDC) and adult mice fed washed Cycas micronesica seed flour develop behavioural and histopathological outcomes that mimic ALS-PDC (Wilson et al., 2002). We have duplicated these initial results in 3-month-old male CD-1 mice, demonstrating a progressive loss of motor function following prolonged cycad feeding. Motor function was measured using the leg extension test and measures of gait length and latency to fall on a wire-hang task. The first, leg extension, measures the integrity of the alpha motor neuron pathway. Each of these tests revealed a significant decrease in behavioral ability in cycad-fed animals. Following cessation of feeding at 100 days, wire-hang and gait length remained abnormal in cycad-fed animals. In contrast, the leg extension function had recovered to normal levels within 2 weeks following the last cycad exposure, remaining constant thereafter. Histological results are still pending, but should provide clues to the nature of the initial behavioural deficit and subsequent recovery. As major neurogenesis is unlikely, the possibility exists that the initial behavioural loss reflects the presence of large numbers of dysfunctional rather than degenerating motor neurons. In this view, the behavioural recovery reflects the recovery of neuronal function. Previous studies using older mice (5-7 mo. old) had not shown a similar recovery of function following cycad exposure (Wilson et al., 2002). Cumulatively, these data may support the notion that the ability to recover from neurological insults is age-dependent involving the restoration of normal function of 'sick' neurons. Support Contributed By: ALSA, SRCF, NSERC, USAMRMC Citation: E.L. Hawkes, J.D. Schulz, C.A. Shaw. RECOVERY OF MOTOR FUNCTION IN MICE FED CYCAD TOXINS: IMPLICATIONS FOR ALS? Program No. 630.11. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. ROLE OF APOE IN NEURODEGENERATIVE DISORDERS USING AN ENVIRONMENTALLY-INDUCED MURINE MODEL OF ALS-PDC. J.M.B.Wilson^1,2*; M.S.Petrik^1; S.C.Grant^5; S.J.Blackband^5; M.Moghadasian^4; C.A.Shaw^1,2,3 1. Opthalmology, 2. Neurosci., 3. Physiology, 4. Hlth.y Heart Program, Univ. of British Columbia, Vancouver, BC, Canada 5. Neurosci., Univ. of Florida, Gainesville, FL, USA Apolipoprotein E (apoE) is the major lipoprotein in the central nervous system and has recently been associated with genetic susceptibility or protection to neurodegenerative diseases. To assess the role of apoE in neurdegenerative disease, a cycad-induced mouse model of amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) was used. Cycad-fed mice have previously displayed progressive cognitive and motor deficits as well as pathological outcomes resembling ALS-PDC (Wilson et al., 2002, 2003). Initial experiments involving apoE include behavioral analysis of mice containing a complete knockout of the APOE gene. The APOE gene product is a lipid transport protein and its absence produces an environment where lipids are not transported to the liver for breakdown and eventual excretion. The identified toxin in cycad is a sterol-glucoside (Khabazian et al., 2002) and a failure to transport it to the brain should result in a decrease in the neurological symptoms seen in our ALS-PDC model. Behavioral data for motor function show that this is the case. ApoE KO mice showed no significant motor deficits but cycad-fed wild-type (WT) mice showed motor deficits. Histology revealed cell death in the substantia nigra, hippocampus, and striatum in cycad-fed WT mice, but not in cycad-fed apoE KO mice. Cycad-fed mice showed increased cholesterol levels without displaying cycad-induced damage to heart or liver. Structural and volume changes in brain and spinal cord were found in cycad-fed WT mice, but not in cycad-fed apoE KO mice. These data show that the apoE KO mice were protected from the effect of the cycad-induced neurodegeneration and support the role of APOE as a possible genetic susceptibility factor for neurodegeneration. Support Contributed By: ALSA, SRCF, NSERC, USAMRMC Citation: J.M.B. Wilson, M.S. Petrik, S.C. Grant, S.J. Blackband, M. Moghadasian, C.A. Shaw. ROLE OF APOE IN NEURODEGENERATIVE DISORDERS USING AN ENVIRONMENTALLY-INDUCED MURINE MODEL OF ALS-PDC. Program No. 630.9. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. THE COMBINED EFFECTS OF CYCAD TOXINS AND THE SOD1 MUTATION ON A MURINE MODEL OF ALS-PDC. C.L.Melder^1; H.B.Bavinton^1; C.Krieger^1*; C.A.Shaw^5 1. Ophthalmology, 2. Physiology, 3. Neurosci., 4. Exptl. Med., Univ. of British Columbia, Vancouver, BC, Canada 5. Kinesiology, Simon Fraser Univ., Burnaby, BC, Canada Both genetic and environmental influences must be considered to gain a comprehensive understanding of the etiology of human neurological disease. Superoxide dismutase (SOD) 1 transgenic mice (B65JL-TGn(SOD1/G93A)1Gur) were behaviourally tested to investigate the known neurodegenerative effects in combination with cycad consumption, know to cause Amyotrophic Lateral Sclerosis-Parkinsonism dementia complex (ALS-PDC). The SOD 1 mutation, up-regulated in 15-20% of human familial ALS, accounts for approximately 1.5% of ALS cases (Mitchell et al., 2000). Male SOD1 and wild-type mice were fed seeds from Cycas micronesica K.D. Hill (hereafter termed cycad) whose consumption is known to cause ALS-PDC in humans (Kurland et al., 1998) and mice (Wilson et al., 2002). Behavioural tests assessed motor and olfactory deficits before and during feeding until sacrifice. Leg extension testing for hind-limb reflexes showed differences among the 4 experimental groups (p<0.005 for all t-tests except wild-type cycad vs. flour) with the sharpest decline seen in the SOD 1 mutant/cycad mice. These data suggest that the effects of the mutation alone are greater than those of the toxin exposure alone, but that there is a compounded deleterious effect of both the mutation and toxin. Rotarod testing of motor coordination resulted in a similar pattern of deficits with the statistically significant exception (p=0.035) of mutant/cycad mice (N=3) performing better than mutant/flour mice (N=1). The present results imply that the combination of environmental and genetic causal factors in neurodegeneration is complex. Further, these data suggest that these 2 factors operate in parallel pathways. Support Contributed By: ALSA, SRCF, NSERC, USAMRMC Citation: C.L. Melder, H.B. Bavinton, C. Krieger, C.A. Shaw. THE COMBINED EFFECTS OF CYCAD TOXINS AND THE SOD1 MUTATION ON A MURINE MODEL OF ALS-PDC. Program No. 630.13. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2003. Online. 2004 DETAILED MAGNETIC RESONANCE MICROSCOPY-DERIVED VOLUME ANALYSIS IN A MOUSE MODEL OF ALS-PDC M.S.Petrik^1,2*; J.M.B.Wilson^2; S.C.Grant^4; S.J.Blackband^4; J.Lai^1; C.A.Shaw^1,2,3 1. Ophthalmology, 2. Neurosci., 3. Exptl. Med., Univ. of British Columbia, Vancouver, BC, Canada 4.Neurosci., Univ. of Florida, Gainesville, FL, USA Cycad (Cycas micronesica) consumption has been shown to induce neurodegeneration in vivo that mimics the progressive neurological disease, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) (Wilson et al., 2002). Previously, specific cortical and subcortical cell loss was measured with stained 2D sections and magnetic resonance microscopy (MRM) was used to measure volumes of affected CNS structures. In this study, MRM was used to further examine neurodegeneration due to cycad consumption. Mice were fed washed cycad as part of their normal diet and showed progressive motor and cognitive behavioral deficits resembling human ALS-PDC. Animals were perfused and intact whole brain and spinal cords were fixed in 4% paraformaldehyde and shipped to the University of Florida. CNS tissue was immersed into 3M^TM Fluorinert^TM and placed into a radio frequency detector coil. T2* scans were performed on CNS tissue using a 750MHZ MRI with a 17.6T magnet. MRM data files were analyzed using Amira 3.1 software to segment out regions of interest and measure volumes of specific brain structures. Resolution of the scans was ~40 m. Cycad-fed mice showed significantly decreased internal capsule/basal ganglia (-39%; *p=0.03) and frontal cortex (-31%; *p=0.05) volumes. Lateral globus pallidus, lateral ventricle, pons, and specific hippocampal areas (CA1, CA2, CA3, dentate gyrus) also showed volume decreases in cycad fed mice but these were not significant. The results of this study reveal additional areas that are affected in our mouse model. These features resemble pathologies seen in human ALS and ALS-PDC. This study serves to further validate our cycad model of ALS-PDC. Support Contributed By: US Army Medical Research and Materiel Command, and the Scottish Rite Charitable Foundation of Canada, NSERC. Citation: M.S. Petrik, J.M.B. Wilson, S.C. Grant, S.J. Blackband, J. Lai, C.A. Shaw. DETAILED MAGNETIC RESONANCE MICROSCOPY-DERIVED VOLUME ANALYSIS IN A MOUSE MODEL OF ALS-PDC Program No. 341.10. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online. DYNAMIC INTERACTION OF GENE AND ENVIRONMENT USING APOE ALLELE VARIANTS IN AN ANIMAL MODEL OF ALS-PDC J.M.B.Wilson^1,2*; M.S.Petrik^1,2; R.L.Rosebrugh^1; M.Moghadasian^4; C.A.Shaw^1,2,3 1. Dept of Opthalmology, 2. Neurosci., 3. Exptl. Med., Univ of British Columbia, Vancouver, BC, Canada 4. Dept of Human Nutr., Univ of Manitoba, Winnipeg, MB, Canada Previous studies have shown progressive behavioral deficits and neuropathological indices in mice fed washed cycad flour (Wilson et al., 2002, 2003), mimicking the human progressive neurological disease, Amyotrophic Lateral Sclerosis parkinsonism dementia complex (ALS-PDC). Recent investigations have shown an interaction between apolipoprotein E (APOE) gene expression and cycad feeding in mice. APOE has also been shown to be a genetic susceptibility locus for Alzheimers disease, ALS, and ALS-PDC and may affect age of onset and disease progression. APOE knock-out (KO) mice fed washed cycad flour displayed no significant motor deficits but showed a partial increase in caspase-3 labeling in the CNS compared to controls; cycad-fed wild-type mice showed significant motor deficits and CNS pathology with abundant caspase-3 labeling in various regions of the CNS compared to controls (Wilson et al., 2004). This current experiment further investigates the interaction between APOE and cycad toxicity. Washed cycad flour was fed to mice expressing the human APOE alleles APOE2, APOE3, and APOE4 and to mice expressing the wild-type mouse APOE gene. Rotarod, wirehang, leg extension, gait length and open field tests were conducted over a 35 week period. Motor tests revealed that all 4 groups of mice showed progressive motor deficits, with APOE2 showing reduced deficits compared to APOE4 and wild-type mice. Cellular analyzes of neuropathology are planned. These results further support the role of APOE as a genetic susceptibility factor in context to a detrimental environmental toxin and may offer some clues to the potential mechanism of cycad toxicity. Support Contributed By: Scottish Rite Charitable Foundation of Canada, NSERC, and the US Army Medical Research and Materiel Command (#DAMD17-02-1-0678) Citation: J.M.B. Wilson, M.S. Petrik, R.L. Rosebrugh, M. Moghadasian, C.A. Shaw. DYNAMIC INTERACTION OF GENE AND ENVIRONMENT USING APOE ALLELE VARIANTS IN AN ANIMAL MODEL OF ALS-PDC Program No. 341.11. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online. SEX DIFFERENCES AND THE ROLE OF OVARIAN HORMONES IN MOTOR DEFICITS AND CELLULAR PATHOLOGY IN A MOUSE MODEL OF ALS-PDC M.C.Wong^1; V.Viau^2; C.A.Shaw^1* 1.Ophthalmology, 2. Anat. and Cell Biol., Univ. of British Columbia, Vancouver, BC, Canada At the peak of its prevalence, Amyotrophic Lateral Sclerosis parkinsonism dementia complex (ALS-PDC) was approximately twice as common in males than in females. Male predominance has also been reported for classical ALS, Parkinsons disease and several other neurodegenerative diseases. These observations have led to the implication of estrogen and progesterone in the protection against neurodegenerative diseases. Although the causal factors of neurodegenerative diseases are generally unknown, ALS-PDC is a neurological disease that has been shown to be highly correlated to the consumption of cycad (Cycas micronesica) flour, which contains several toxins. We developed an animal model of cycad-induced ALS-PDC that mimics several features in humans, including gait, balance and reflex disturbances. We found that the severity of motor deficits was greater in ovariectomized than in hormone-replaced and intact female mice. Interestingly, these deficits were most severe in male mice. To investigate if these motor effects are accompanied by changes in CNS cell turnover and/or damage, we examined cell proliferation using BrdU and apoptosis characterized with activated caspase 3 antisera and TUNEL. Preliminary analyses of the motor cortex, spinal cord and striatum suggest that overall, there is increased cell proliferation and apoptotic cell death in males and ovariectomized females relative to intact and hormone-replaced female mice. The combined behavioral and histological data strongly suggest that ovarian hormones play a protective role against neurotoxins and that the differential impact may partially explain the sex differences found in several neurodegenerative diseases. Support Contributed By: Scottish Rite Charitable Foundation of Canada, US Army Medical Research and Materiel Command DAMD17-02-1-0678 (to CAS) Citation: M.C. Wong, V. Viau, C.A. Shaw. SEX DIFFERENCES AND THE ROLE OF OVARIAN HORMONES IN MOTOR DEFICITS AND CELLULAR PATHOLOGY IN A MOUSE MODEL OF ALS-PDC Program No. 341.12. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online. 2005 Comparative study of the roles of novel cycad toxins versus BMAA in the etiology of ALS-PDC Reyniel Cruz-Aguado^a,b, Jeff Schulz^a,b,c, Erin Hawkes^a,b,c, Swaraj Singh^a,b, and Christopher A. Shaw^a,b,c 1.Department of Ophthalmology^a, Neural Dynamics Research Group^b and Neuroscience Program^b, University of British Columbia, 828 W.10^th Ave., Vancouver, BC, Canada V5Z 1L8 Amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC) is a neurological variant found on Guam, wherein symptoms of the three major neurodegenerative disorders can co-occur. The neurotoxins contained in cycad seeds, a food staple in Guam, have been proposed as a causal environmental agent. Some investigators support the notion that a plant amino acid, ß-methyl-L-aminoalanine (BMAA) is the causal neurotoxin. In order to compare the unknown toxins in cycad vs. BMAA, we fed mice with cycad flour or BMAA-containing chow, and conducted a set of neurobehavioral tests of motor and cognitive function (rotarod, wire hang, clasping reflex, stride length, water maze). The brains of control, cycad-fed, and BMAA-fed mice were processed for the analysis of cell death, neurotransmitter-synthesizing enzymes and oxidative markers. Mice fed with cycad flour showed behavioral and morphobiochemical outcomes that are consistent with the symptoms and neuropathological pattern of ALS-PDC. The present findings show that cycad exposure induces a region-specific pattern of oxidative damage. In addition, we found a differential vulnerability of the neuronal phenotypes towards cycad toxicity. Cycad-fed animals, compared with age-matched controls, show a higher level of oxidized DNA in the CA1 hippocampal region and also a higher content of protein oxidative products in the striatum. BMAA failed to induce neuropathological changes in mice, which may be explained by the poor brain-blood barrier permeability of this amino acid. These findings do not support the role of BMAA in the etiology of the Gambian ALS-PDC, and suggest that other toxins present in washed cycad flour might be the causal factor. In addition, these results demonstrate that an increased generation of oxidant molecules is a component of the cycad toxin spectrum of actions. Supported by: US Army Medical Research and Materiel Command IS THERE A CAUSAL ROLE OF STERYL GLUCOSIDE NEUROTOXINS IN ALS? Chung, JS, Wilson JMB, Hawkes, EL, Cudkowicz, ME, Newhall, K, Brown, RH, Shaw, CA. The causal factor(s) responsible for sporadic neurological diseases are unknown and the stages of disease progression remain undefined and poorly understood. Epidemiological studies of the Guamanian variant of ALS, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) have shown a positive correlation between consumption of cycad seed flour and the development of the disease. In vivo studies in which adult male mice consume cycad seed flour as part of diet show that treated animals show profound and progressive motor, cognitive, and olfactory behavioural deficits combined with the loss of neurons in each of the respective neural subsets. The expression of these outcomes mirrors precisely the behavioural and pathological deficits in ALS-PDC. In vitro experiments using isolated cycad fractions have identified the likely toxins as variant steryl glucoside molecules contained in cycad flour, specifically b-sitosterol b-D-glucoside (BSSG), campesterol b-D-glucoside, and stigmasterol b-D-glucoside) are neurotoxic. Preliminary in vivo experiments with mice fed BSSG as part of their diet for 10 weeks show a significant 25% loss of motor neurons in the lumbar spinal cord with increased caspase-3 labelling. We have begun screening for these putative human ALS and control patients. HPTLC was used to measure steryl glucoside concentration in 40 ALS and 30 control plasma samples. Initial results show increased steryl glucoside concentrations in ALS patients. The identification of steryl glucosides as neurotoxins in the animal model and the preliminary data suggesting elevated levels of these molecules in human neurological disease warrants further investigation into further studies of steryl glucoside as causal neurotoxins or as biomarkers of disease status or progression. IMPLICATIONS OF VACCINE TOXICITY OF ALS-GWS: BEHAVIOURAL AND PHYSIOLOGICAL EFFECTS OF ALUMINUM HYDROXIDE AND SQUALENE ADJUVANTS IN MICE. M.S. Petrik^1,2, R.F. Garry^5, R. C. Tabata^1, J. Chung^1, M.C. Wong^1,2, A. Villaruel^1, M. Nass^6,and C.A. Shaw^1,3,4 ^1Departments of Ophthalmology, ^3Physiology, and ^4Experimental Medicine ^2Program in Neuroscience, University of British Columbia, Vancouver, British Columbia, Canada; ^5Department of Microbiology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA; ^6Mount Desert Island Hospital, Bar Harbor, Maine, USA. Gulf War Syndrome is a multisystemic illness of uncertain origin that affected many veterans of the 1991 conflict. A significant number of patients with GWS exhibit symptoms of ALS including fatigue, memory impairment, confusion, muscle pain, and motor function loss. Various adjuvants contained in the anthrax vaccine administered to deployed troops have been implicated with ALS-GWS, notably aluminum hydroxide and possibly squalene. In an attempt to model the disease, adult male CD-1 mice received two injections (two weeks apart) of aluminum hydroxide (50µl) and squalene (40µl) adjuvants. Control mice were injected with 0.9% PBS. Mice were then subjected to several behavioural tests including rotorod, wirehang, open field, paw print, and water maze over a period of six months. Blood samples were analyzed during the test period and at the time of sacrifice for the presence of squalene antibodies using an anti-squalene antibody assay. Histology was performed to examine CNS tissue for evidence of neurodegeneration. Mice injected with aluminum hydroxide alone showed loss of muscle strength, abnormal weight gain, increased anxiety levels and the presence of apoptotic neurons in lumbar spinal cord. Mice injected with squalene showed increased anxiety levels, apoptotic neurons and presence of squalene antibodies. Mice injected with aluminum hydroxide and squalene showed the presence of apoptotic neurons and squalene antibodies. Anthrax vaccine adjuvants appear to have negative impacts on motor behaviour and neuron survival and give outcomes similar to those seen in ALS-GWS. These results suggest that further studies of possible toxicity of these compounds are warranted. Supported by: US Army Medical Research and Materiel Command, and the Scottish Rite Charitable Foundation of Canada, NSERC. COMPARISON OF GENETIC AND ENVIORNMENTAL MOUSE MODELS OF ALS USING MAGNETIC RESONANCE MICROSCOPY AND HISTOCHEMISTRY Petrik MS^1, 2, Wilson JMB^1, 2, Grant SC^3, Blackband SJ^3, Shan X^4, Schulz JD^1, 2, Singh S^2, 6, Krieger C^4, 7and Shaw CA^1, 2, 5, 6 Program in Neuroscience^1, Departs. of Ophthalmology^2, Physiology^5, and Experimental Medicine^6, Faculty of Medicine^7, University of British ColumbiaVancouver, BC, Canada; Depart. of Kinesiology^4, Simon Fraser University, Burnaby, BC, Canada; Depart. of Neuroscience^3, University of Florida, Gainesville, FL, USA. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting spinal motor neurons and descending motor tracts of the central nervous system. To compare murine models that mimic the SOD form of familial ALS to an environmental model based on ALS-parkinsonism dementia complex (ALS-PDC), we evaluated lumbar spinal cords (SC) from mice over-expressing human mutant superoxide dismutase (mSOD) and cycad-fed mice. Spinal cord tissue was compared using magnetic resonance microscopy (MRM). Three-dimensional analysis of SC volumes was performed from T2* microimages acquired at 40mm using a 17.6-T magnet. mSOD mice showed significantly decreased volumes in total grey (-15%) and white matter (-23%) of SC segments, compared to controls; ventral horn volumes in mSOD mice showed a -10% decrease in volume but no significant change in dorsal horn. Mice fed cycad seed flour have previously been shown to mimic all of the essential features of ALS-PDC. Cycad-fed mice showed significantly decreased volumes in SC grey matter (-21%), with all of the observed changes in the ventral horn. No significant changes in total white matter volume were observed. Histological assessment revealed that both models had significantly reduced motor neuron numbers and showed increased astrogliosis and microglial proliferation. These results indicate major differences in the extent of white matter involvement in two murine models of ALS that reflect variation in the extent of degeneration in descending tracts. The dissimilarity in the pathogenesis and overall behavioural deficits in the two ALS models may suggest differerences in the type of ALS that these models represent. These data also highlight the utility of MRM in the assessment neurological disease. Supported by: US Army Medical Research and Materiel Command, and the Scottish Rite Charitable Foundation of Canada, NSERC. THE PROGRESSION OF MOTOR DEFICITS AND CNS PATHOLOGY IN ALS-PDC JMB Wilson^1, MS Petrik^1, M Moghadasian^4, SC Grant^5, S Blackband^5, C Krieger^6,7, and UK Craig^8, CA Shaw^1,2,3 ^1Neuroscience Program and Depts. of ^2Ophthamology and ^3Experimental Medicine, ^7Faculty of Medicine, University of British Columbia, Vancouver BC, Canada; ^4Depts. of Human Nutritional Sciences and Pathology, University of Manitoba, Winnipeg, MB, Canada; ^5Dept. of Neuroscience, University of Florida, Gainesville, USA; ^6Dept. of Kinesiology, Simon Fraser University, Burnaby, BC, Canada; ^8Bodig and Lytico Research Project, University of Guam, Mangilao, Guam. Epidemiological studies have shown a positive correlation between cycad flour consumption and the development of the neurodegenerative disorder, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) of the Western Pacific. The presumed causal factor(s) is considered to be one or more of the toxins contained in cycad seed flour. Epidemiological data also suggest a correlation of ALS-PDC and particular susceptibility genes such as those coding for Apolipoprotein E (ApoE). In vivo studies of mice fed washed cycad seed flour and isolated cycad toxins were tested with a battery of behavioural measures. Magnetic resonance microscopy (MRM) scans of the brains and spinal cords of cycad treated animals and conventional histology were conducted to determine the level of CNS pathology of cycad fed mice. In addition, several genetically altered ApoE mouse strains were fed cycad to examine the interaction between environmental toxicity and genetic susceptibility. The behavioural testing showed a temporal sequence of deficits that correlated in a non-linear manner to neural cell death in the CNS. MRM studies and histology showed decreased volumes in the CNS and CNS pathology similar to that seen human ALS-PDC. Experiments with ApoE mice variants revealed altered susceptibility to cycad toxins and differing rates of disease progressions. Onset of behavioural deficits was preceded by significant CNS pathology with ALS-like deficits occurring prior to Parkinsonism features, suggesting a temporal sequence of neurodegeneration due to cycad toxicity. Supported by: US Army Medical Research and Materiel Command, and the Scottish Rite Charitable Foundation of Canada, NSERC. Keywords: ALS, Parkinson’s disease, Dementia, ALS-PDC, MRM, Cycad, APOE. EFFECTS OF INHIBITION OF MICROGLIAL ACTIVATION IN A MURINE MODEL OF ALS-PDC S.Singh^1,2*; P.T.T.Ly^1,2; A.Khurana^1; C.A.Shaw^1,2,3 1.Ophthalmology, 2. Experimental Medicine, 3. Program in Neuroscience, Univ. of British Columbia, Vancouver, BC, Canada We have developed an animal model of amyotrophic lateral sclerosis-parkinsonism dementia complex which mimics the essential features of the disease with the initial neurological insult arising from neurotoxin(s)in washed cycad seeds. Accumulating evidence suggests a key role of microglial activation in ALS and Parkinsons disease. This led us to hypothesize that cycad exposure may cause microglial activation and lead to disease manifestation. Minocycline (Mc) was used to block microglial activation. Three groups of CD1 male mice, 4 months old were used. Control group was fed a normal diet. Cycad group received cycad flour mixed with normal diet. Cycad/Mc group had the same diet as cycad group. Once cycad-fed animals showed neurobehavioral impairment, Mc (1mg/g BW) was added to the diet of the last group. We followed symptoms of motor neuron degeneration measured by four different behavioral tests for three months, after which brains and spinal cords from the animals were prepared for histology. Both cycad groups showed deterioration in three behavioral tests. Mc-treated animals showed a recovery in two behavioral tests. The histological analysis of lumbar spinal cords, cycad fed animals had significantly lower motor neuron count, higher amounts of microglial activation (OX6), inflammation (p38MAPK), astrogliosis (GFAP) and apoptosis (activated caspase-3) compared to control animals. Mc/cycad fed animals had a higher motor neuron count compared to cycad animals, but neurons appeared atrophic. Mc/cycad fed animals also had less microglial activation, inflammation, astrogliosis, and apoptosis compared to cycad fed animals. Compared to controls, Mc/cycad fed animals had higher number of apoptotic cells. In conclusion Mc/cycad fed animals demonstrated a partial recovery. This suggests that either microglial activation is an early event in neurodegeneration or that cycad exposure activates other cellular pathways. Support Contributed By: US Army Medical Research and Material Command. Citation S. Singh, P.T.T. Ly, A. Khurana, C.A. Shaw. EFFECTS OF INHIBITION OF MICROGLIAL ACTIVATION IN A MURINE MODEL OF ALS-PDC Program No. 332.4. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2005. Online. 2006 Neurotoxic susceptibility in a mouse model of ALS2 as revealed by behavioural analysis G. Lee, R. Cruz-Aguado, P.C. Orban, T.G. Hampton, S. McCue, M.R. Hayden, C.A. Shaw Loss-of-function mutations of the alsin gene (Als2) are related to a juvenile-onset form of motor neuron disease known as ALS2. Alsin is involved in the regulation of guanine nucleotide signalling pathways. Although knock-out Als2^-/- mice show subtle behavioural alterations, they do not fully exhibit the neurological deficits observed in individuals with ALS2. This raises the possibility that the ALS2 phenotype is attributed to the interaction of genetic and environmental factors. Therefore, we studied the influence of the dietary exposure to cycad seeds on the motor performance of Als2^-/- and wild-type mice hypothesizing that Als2^-/- mice are possibly more susceptible to neurotoxic insults. The neurotoxins in cycad seeds have been proposed as a causative environmental agent of the amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC), a neurological syndrome clustered in Guam and other locations in the Western Pacific. Als2^-/- and wild-type mice were fed cycad flour as part of their normal diet (0.5 g cycad flour/day during 6 months) and subjected weekly to motor function tests including rotarod, wire hang, clasping reflex, stride length, and beam walking. A group of wild-type cycad-fed animals showed significant motor neuron loss in the lumbar spinal cord. No significant differences in motor function between knock-out and wild-type mice were observed using these tests. At the end of the feeding period, a detailed gait analysis was performed using ventral plane videography (DigiGait). Als2^-/- mice showed a more athletic gait by a smaller hind limb splay angle (p < 0.05) and a less variable stance width (p < 0.05). The cause of this supernormal gait performance in alsin knock-out mice is unclear, but a similar phenomenon has been observed in the SOD G93A genetic model of ALS. This may represent an over compensatory response of the motor system to an underlying pre-symptomatic neurodegenerative event. In conclusion, our findings suggest that Als2^-/- mice exposed to cycad toxins do not show an obvious behavioural impairment as compared to wild-type animals. These data underscore the complexity of the gene-environment interaction in neurodegenerative diseases. The neurotoxic effects of b-sitosterol glucosides in NSC 34 cells, a mouse motor neuron-derived cell line P. T.T Ly, X.B. Liang, Q. Wang, K. Andreasson, and Christopher A. Shaw There is increasing evidence that suggests environmental toxins are involved in the pathogenesis of neurodegenerative diseases. Epidemiological studies have correlated the cluster of the Guamanian amyotrophic lateral sclerosis (ALS-PDC) to consumption of seeds from the cycad. Cell death assays using different fractions from cycad seeds suggested that sterol glucosides are one of the possible neurotoxic components. This study investigates the in vitro neurotoxic effects of a synthetic sterol glucoside, b-sitosterol glucosides (BSSG). We used NSC34 cells, a cell line derived from the fusion of mouse spinal cord motor neuron cells with neuroblastoma, and spinal cord organotypic cultures. NSC34 cells treated with increasing doses (0, 0.1, 1, 5, 10, 25, 50 mM) of synthetic BSSG for 5 days show a decrease in cell viability as determined by the trypan blue dye exclusion test (one way ANOVA, p<0.001). Likewise, data from postnatal rat spinal cord organotypic culture slices treated with BSSG for 3 weeks show significant motor neuron loss as compared to the controls. In order to explore the cellular pathways involved in the neurotoxic actions of BSSG, we used immunohistochemical analysis of proteins involved in neuronal death. The analysis revealed that NSC34 cells treated with 50 μM BSSG have significantly increased expression of the cell stress protein HSP70 and the activated form of the pro-apoptotic caspase 3 (two-tail Student’s t test, p<0.05). Furthermore, BSSG treatment induces a dose-dependent decreased of the heavy subunit of neurofilament (NFH) expression (two tail Student’s t test, p<0.01), suggesting disruption of the cytoskeleton as an event in cellular degeneration. A similar decrease in NFH has been observed in other in vitro models of neurodegeneration. In conclusion, our study confirms that BSSG is a bioactive agent in the seeds of cycad and may be a neurotoxin involved in neurodegenerative events. Supported by: US Army Medical Research and Materiel Command Aluminum adjuvant linked to Gulf War Illness induces motor neuron death and neural inflammation in mouse CNS CA Shaw^1,, MS Petrik^1,2, MC Wong^1,2, and RC Tabata^3 Depts. of Ophthalmology^1, and Experimental Medicine^3, Program in Neuroscience^2, University of British Columbia, Vancouver, BC, Canada Gulf War Illness (GWI), popularly termed Gulf War Syndrome (GWS) is a multisystemic illness of unknown origin that affects many veterans of the 1991 conflict. A significant number of patients with GWI exhibit symptoms consistent with neurological dysfunction, including fatigue, memory impairment, confusion, muscle pain, and motor function loss. A form of ALS associated with GWI is present at rates roughly twice the national average at significantly younger ages. Much attention has focused on the possible role of the anthrax vaccine (AVA) with a particular focus on various adjuvants, notably aluminum hydroxide and squalene. Aluminum salts of various kinds are widely used in a number of vaccines. Squalene has been used in some vaccines licensed outside the United States. The manufacturers of AVA deny that squalene was ever part of vaccine formulation, although squalene antibodies are preferentially found in GWI patients (Asa et al., 2002). To examine whether these adjuvants contribute to neurological outcomes associated with GWI, we injected young male colony CD-1 mice with either compound alone or in combination at doses (wt/wt) equivalent to those given to service personnel. Adult male CD-1 mice received two injections (two weeks apart) of aluminum hydroxide (50µg/kg), squalene (2% suspension), or a combination. Control mice were injected with 0.9% PBS. Mice were then subjected to behavioral tests of motor and cognitive function over a period of six months. Following sacrifice, CNS tissues were examined using immunohistochemistry for evidence of neural death and inflammation. Significant motor function losses were found in the aluminum group and significant cognitive deficits appeared in the combined group. Significant increases in apoptotic neurons were found in lumbar spinal cord, red nucleus, and motor cortex following aluminum injection. This same treatment group showed significant motor neuron loss and increases in GFAP positive astrocytes and Iba-1 positive microglia in the lumbar spinal cord. Our findings demonstrate a possible role for AVA adjuvants in some neurological features associated with GWI. The data further suggest that additional studies are needed to fully evaluate the potential impact of such vaccine adjuvants on the general population. Supported by: Scottish Rite Charitable Fdn. of Canada and NSERC Canada. In Vivo Sterol Glucoside Neurotoxicity: Implications for ALS-PDC and ALS JMB Wilson, RC Tabata, CA Shaw The causal factor(s) responsible for sporadic neurological diseases are unknown and the stages of disease progression remain undefined and poorly understood. Epidemiological studies of the Guamanian variant of ALS, ALS-PDC, have shown a positive correlation between consumption of washed cycad seed flour and the development of the disease. In vitro experiments using isolated cycad fractions have identified some of the likely neurotoxins as sterol glucoside molecules contained in washed cycad flour. The present study aims to asses the role of sterol glucoside molecules as causal agents in ALS-PDC. In vivo studies in which b-sitosterol b-D glucoside (BSSG) was fed to mice as part of their diet for 10 weeks were conducted. Mice were tested using an established battery of behavioural tests and histological methods previously used in cycad-fed mice. Mice fed BSSG show a significant loss of motor neurons in the lumbar spinal cord (30-45% loss) and increased labelling for the cell stress marker ATF-3. This motor neuron loss appears to be progressive as cell loss continued even after cessation of BSSG feeding. These data compliment in vitro data showing BSSG induces cell death, cytoskeletal disruption and increased HSP-70 expression in a motor neuron-derived cell line (P. Ly) and motor neuron loss in an organotypic spinal cord preparation (K. Andreasson). The identification of sterol glucosides as neurotoxins in the animal model warrants further investigation into further studies of sterol glucoside as causal neurotoxins or as biomarkers of disease status or progression. Supported by: Scottish Rite Charitable Fdn. of Canada, NSERC, and US Army Medical Research and Material Command Environmentally-induced parkinsonism in cycad-fed rats. K. M. Valentino^1, N. V. Dugger^2, E. Peterson^3, J. M. Wilson^4, C. A. Shaw^4,5, *P. J. Yarowsky^1,2; ^1Dept Pharmacol & Exp Therapeut, Univ Maryland Sch Med, Baltimore, MD, ^2Dept of Research, VA Maryland Healthcare System, Baltimore, MD, ^3Genetic Counseling Program, Univ Maryland Sch Med, Baltimore, MD, ^4Depts of Opthalmology and Neuroscience Programme, Univ of British Columbia, Vancouver, BC, CANADA, ^5Dept of Physiol, Univ of British Columbia, Vancouver, BC, CANADA. Previous epidemiological studies of the Guamanian variant of ALS-PDC (Amyotrophic lateral sclerosis-Parkinsonism dementia complex) have demonstrated a causative link based on the consumption of cycad seed flour indigenous to the area. In cycad-fed mice, significant CNS pathology and behavior deficits were observed similar to the neurodegenerative symptoms of ALS (Wilson, et. al., 2002). In our current study, we fed eight (~90 days old) Sprague -Dawley male rats for 23 weeks with washed cycad seed flour. Eight control rats were fed kitchen baking flour. Several behavioral tests were administered and video-recorded during the feeding time frame and are still continuing on rats not yet sacrificed for histological study. All cycad-fed rats displayed Parkinsonism-like behaviors. Unlike the previously published mice cohort (Wilson, et. al., 2002), 7/8 cycad-fed rats began to display spontaneous home cage rotations after 12 weeks of feeding. Parkinson’s disease can be identified by bilateral or unilateral lesions in the Substantia Nigra pars compacta (SNpc) of the brain. It was, our assumption, that the cycad-fed rats that spontaneously rotated may have a unilateral SN defect. One cycad-fed rat in our study, which never was observed to rotate, displayed a “sleepy” state during home cage and behavioral testing observations. Sessions with Apomorphine “woke-up” this rat and in addition, caused the other cycad- fed rats to show other dyskinetic motions and hyperactivity (Jinnah, 1991). This lends evidence to more of a Parkinsonism state than ALS. At this time, there has been no paralysis observed as associated with ALS. Two rats from the cycad and flour group each were sacrificed and processed for histology. Preliminary results show a unilateral SN defect characterized by the losses of tyrosine hydroxylase and neuron number, as well as increases in ubiquitin and alpha-synuclein in this area in the affected brain side. Our study not only shows another paradigm for cycad as an environmental neurotoxin but also may be able to be used in future Parkinson’s disease studies.