Chronic exposure to dietary sterol glucosides is neurotoxic to motor neurons and induces an ALS-PDC phenotype.

Epidemiological studies of the Guamanian variants of amyotrophic lateral sclerosis (ALS) and parkinsonism, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC), have shown a positive correlation between consumption of washed cycad seed flour and disease occurrence. Previous in vivo studies by our group have shown that the same seed flour induces ALS and PDC phenotypes in out bred adult male mice. In vitro studies using isolated cycad compounds have also demonstrated that several of these are neurotoxic, specifically, a number of water insoluble phytosterol glucosides of which beta-sitosterol beta-D: -glucoside (BSSG) forms the largest fraction. BSSG is neurotoxic to motor neurons and other neuronal populations in culture. The present study shows that an in vitro hybrid motor neuron (NSC-34) culture treated with BSSG undergoes a dose-dependent cell loss. Surviving cells show increased expression of HSP70, decreased cytosolic heavy neurofilament expression, and have various morphological abnormalities. CD-1 mice fed mouse chow pellets containing BSSG for 15 weeks showed motor deficits and motor neuron loss in the lumbar and thoracic spinal cord, along with decreased glutamate transporter labelling, and increased glial fibrillary acid protein reactivity. Other pathological outcomes included increased caspase-3 labelling in the striatum and decreased tyrosine-hydroxylase labelling in the striatum and substantia nigra. C57BL/6 mice fed BSSG-treated pellets for 10 weeks exhibited progressive loss of motor neurons in the lumbar spinal cord that continued to worsen even after the BSSG exposure ended. These results provide further support implicating sterol glucosides as one potential causal factor in the motor neuron pathology previously associated with cycad consumption and ALS-PDC.

Magnetic resonance microscopy and immunohistochemistry of the CNS of the mutant SOD murine model of ALS reveals widespread neural deficits.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons and descending motor tracts of the CNS. We have evaluated the CNS of a murine model of familial ALS based on the over-expression of mutant human superoxide dismutase (mSOD; G93A) using magnetic resonance microscopy (MRM) and immunohistochemistry (IHC). Three-dimensional volumetric analysis was performed from 3D T2*-weighted images acquired at 17.6 T at isotropic resolutions of 40 mum. Compared to controls, mSOD mice had significant reductions in the volumes of total brain, substantia nigra, striatum, hippocampus, and internal capsule, with decreased cortical thickness in primary motor and somatosensory cortices. In the spinal cord, mSOD mice had significantly decreased volume of both the total grey and white matter; in the latter case, the volume change was confined to the dorsal white matter. Increased apoptosis, GFAP positive astrocytes, and/or activated microglia were observed in all those CNS regions that showed volume loss except for the hippocampus. The MRM findings in mSOD over-expressing mice are similar to data previously obtained from a model of ALS-parkinsonism dementia complex (ALS-PDC), in which neural damage occurred following a diet of washed cycad flour containing various neurotoxins. The primary difference between the two models involves a significantly greater decrease in spinal cord white matter volume in mSOD mice, perhaps reflecting variations in degeneration of the descending motor tracts. The extent to which several CNS structures are impacted in both murine models of ALS argues for a reevaluation of the nature of the pathogenesis of ALS since CNS structures involved in Parkinson's and Alzheimer's diseases appear to be affected as well.

Late appearance of glutamate transporter defects in a murine model of ALS-parkinsonism dementia complex.

Excitotoxicity has been widely hypothesized to play a major role in various neurodegenerative diseases. We have used a mouse model of ALS-parkinsonism dementia complex (ALS-PDC) of the Western Pacific to explore this hypothesis. Mice fed washed cycad flour, the major epidemiological link to ALS-PDC, showed significant and progressive motor, cognitive, and sensory behavioural deficits [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207-221]. In addition, glutamate transporter (GLT-1/EAAT2) levels measured by immunohistochemistry with antibodies specific for two glial glutamate transporter splice variants (GLT-1alpha and GLT-1B) were significantly down-regulated showing a 'patchy' loss of antibody label centered on blood vessels [Wilson, J.M., Khabazian, I., Pow, D.V., Craig, U.K., Shaw, C.A., 2003. Decrease in glial glutamate transporter variants and excitatory amino acid receptor down-regulation in a murine model of ALS-PDC. Neuromol. Med. 3 (2), 105-118]. Receptor binding assays showed decreased NMDA and AMPA receptor levels combined with increased GABA(A) receptor levels in various CNS regions. The alterations in GLT-1 variants and the ionotropic receptors are consistent with an increased level of extracellular glutamate. The interaction between environmental toxicity and genetic susceptibility was also tested using mice expressing various Apolipoprotein E (ApoE) genotypes. Mice lacking the ApoE gene showed relative resistance to cycad-induced toxicity as measured by GLT-1B labeling, but all mice expressing the human ApoE isoforms showed a similar loss of GLT-1B. We have further shown that an isolated cycad toxin (beta-sitosterol-beta-d-glucoside, BSSG), previously shown to release glutamate in vitro [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207-221], can be directly toxic to motor neurons in vivo [Wilson, J.M., Petrik, M.S., Moghadasian, M.H., Shaw, C.A., 2005. Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC. Can. J. Physiol. Pharmacol. 83 (2), 131-141]. However, BSSG-fed mice did not show altered GLT-1B labeling in the spinal cord suggesting that an initial excitotoxic mechanism may not be responsible for the final neuronal loss observed. While glutamate-mediated excitotoxicity is likely involved in the outcomes following cycad/BSSG exposure, the precise location in the cascade of events ultimately leading to neuronal death remains to be determined.

Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC.

Epidemiological studies have shown a positive relationship between cycad flour consumption and the development of the neurodegenerative disorder, amyotrophic lateral sclerosis - parkinsonism - dementia complex (ALS-PDC). Apolipoprotein E (apo E) allele variations have been associated with genetic susceptibility in neurodegenerative diseases, including ALS-PDC. We have studied cycad toxicity in a mouse model of ALS-PDC with a particular interest in its impact on the central nervous system (CNS) in both apo E knock-out (KO) mice and their wild-type (WT) counterparts. Behavioral motor tests, motor neuron counts, and immunohistochemical staining in brain and spinal cord, as well as routine histological examinations on internal organs, were performed to evaluate cycad toxicity. Plasma cholesterol levels were also measured before and during the study. Cycad treatment was associated with higher levels of plasma cholesterol only in apo E KO mice; increased levels of plasma cholesterol did not result in increased athero genesis. Cycad-fed wild-type mice developed progressive behavioral deficits including ALS-PDC-like pathological outcomes, while cycad-fed apo E KO mice were not significantly affected. Cycad-fed wild-type mice had shorter gait length measurements along with higher active caspase-3 levels in the striatum, substantia nigra, primary motor cortex, and spinal cord as compared with corresponding controls. These changes were associated with decreased labeling for glutamate transporter 1B and tyrosine hydroxylase activity levels. No evidence of cycad toxicity was observed in internal organs of either wild-type or apo E KO mice. Our data demonstrate that apo E KO mice are less susceptible to cycad toxicity, suggesting a role for apo E as a possible genetic susceptibility factor for some forms of toxin-induced neurodegeneration.

Quantitative measurement of neurodegeneration in an ALS-PDC model using MR microscopy.

Exposure to cycad (Cycas micronesica K.D. Hill) toxins via diet has been shown to induce neurodegeneration in vivo that mimics the progressive neurological disease, amyotrophic lateral sclerosis--parkinsonism dementia complex (ALS--PDC). In previous studies, specific cortical and subcortical cell loss was measured with conventional stained sections. In the present study, magnetic resonance (MR) microscopy was used to examine neurodegeneration in three dimensions (3D) in isolated intact brains and spinal cords. Mice were fed washed cycad for 2 months and showed progressive motor deficits resembling human ALS--PDC. CNS tissue was imaged at 17.6 T. T2* scans were acquired on both spinal cord and brain samples with an isotropic resolution of 41 microm. Through MR volumetrics, cycad-fed mice showed significantly decreased volumes in lumbar spinal cord gray matter, substantia nigra, striatum, basal nucleus/internal capsule, and olfactory bulb. Cortical measurements of conventionally stained sections revealed that cycad-fed mice also showed decreased cortical thickness. These results show that MR microscopy (MRM) is sensitive enough to measure degeneration in this early stage model of a progressive neurological disease with strong correlations to behavioral deficits and histological results and may be applicable in vivo to the same model. Similar analysis may be used in the future as a diagnostic aid in tracking the early progression of neurological disorders in preclinical human subjects.

Analysis of neurological disease in four dimensions: insight from ALS-PDC epidemiology and animal models.

The causal factor(s) responsible for sporadic neurological diseases are unknown and the stages of disease progression remain undefined and poorly understood. We have developed an animal model of amyotrophic lateral sclerosis-parkinsonism dementia complex which mimics all the essential features of the disease with the initial neurological insult arising from neurotoxins contained in washed cycad seeds. Animals fed washed cycad develop deficits in motor, cognitive, and sensory behaviors that correlate with the loss of neurons in specific regions of the central nervous system. The ability to recreate the disease by exposure to cycad allows us to extend the model in multiple dimensions by analyzing behavioral, cellular, and biochemical changes over time. In addition, the ability to induce toxin-based neurodegeneration allows us to probe the interactions between genetic and epigenetic factors. Our results show that the impact of both genetic causal and susceptibility factors with the cycad neurotoxins are complex. The article describes the features of the model and suggests ways that our understanding of cycad-induced neurodegeneration can be used to decipher and identify the early events in various human neurological diseases.

Isolation of various forms of sterol beta-D-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS-parkinsonism dementia complex.

The factors responsible for ALS-parkinsonism dementia complex (ALS-PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin-linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non-water soluble compounds from washed cycad flour and have identified three sterol beta-d-glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non-toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT-mediated biotin-dUTP nick-end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase-3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS-PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.