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Neuroscience and Biobehavioral Reviews xx (2003) xxxxxx www.elsevier.com/locate/neubiorev

Review

Analysis of neurological disease in four dimensions: insight from ALS-PDC epidemiology and animal models
C.A. Shawa,b,c,d,*,1, J.M.B. Wilsona,1
a Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada Department of Ophthalmology, University of British Columbia, Vancouver, BC, Canada c Department of Physiology, University of British Columbia, Vancouver, BC, Canada d Department of Experimental Medicine, University of British Columbia, Vancouver, BC, Canada b

Received 1 May 2003; revised 9 July 2003; accepted 14 August 2003

Abstract The causal factor(s) responsible for sporadic neurological diseases are unknown and the stages of disease progression remain undened 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 decits in motor, cognitive, and sensory behaviors that correlate with the loss of neurons in specic 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. q 2003 Published by Elsevier Ltd.
Keywords: Amyotrophic lateral sclerosis-Parkinsonism dementia complex; Alzheimers disease; Parkinsonism; Amyotrophic lateral sclerosis; Cycad; Neurodegeneration; Excitotoxicity; Sterol glucoside; Animal model; Time course

Contents 1. Introduction: the fundamental problems in neurological disease research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Dening neurological disease in four dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Timeline concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Overview of age-related neurological diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Is there a neurological Rosetta Stone?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. A murine model of ALS-PDC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Does the ALS-PDC model satisfy standard criteria? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Genetic epigenetic interactions in the cycad model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Time line of neurodegenerative events in the cycad model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Template matching to human neurological disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The mouse model of ALS-PDC: implications for prophylaxis and for halting disease progression . . . . . . . . . . . . . . 12. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Address: Research Pavilion, VGH, Rm 386, 828 West 10th Ave., Vancouver, BC, Canada V6T 1Z3. Tel.: 1-604-8754111x68375; fax: 1-604-875-4376. E-mail address: cshaw@interchange.ubc.ca (C.A. Shaw). 1 Equal co-authors. 0149-7634/$ - see front matter q 2003 Published by Elsevier Ltd. doi:10.1016/j.neubiorev.2003.08.001

1. Introduction: the fundamental problems in neurological disease research Of all the diseases that humans have sought to understand and thereby control, those involving the nervous system have

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proven to be the most intractable. Within the realm of neurological diseases, those that are age-dependent (i.e. Alzheimers Disease (AD), Parkinsons Disease (PD) and amyotrophic lateral sclerosis (ALS)) are the most common and the most difcult to track in time given that they may be initiated early in life yet normally express in late middle to old age. For these diseases, there are neither denitive hypotheses nor unambiguous experimental data showing what the causal factors are likely to be. In particular, we are still uncertain whether these diseases have genetic and/or environmental origins and how factors associated with either can inuence neural cell survival. It is also unclear how pathological processes evolve from the initial insult(s) through to cell death, or what the functional relationships are between neural pathology and behavioral outcome at any stage of the disease process. Postmortem studies have identied numerous molecules that appear to be altered in amount or function, but the larger context or interrelationships between such molecules, and the temporal sequence of changes in the affected cells or neural systems are largely unknown. Details about rates of progression from clinical diagnosis to death are known, but minimal details are available about early preclinical events before classical symptoms are observed. Therapeutic intervention has proven to be difcult due to the lack of a clearly established link to any of the putative causal factors for neurological disease. The prevention of disease initiation becomes challenging without knowing in advance which individuals are susceptible and for what reasons. Similarly, without understanding or being able to identify pre-clinical symptoms, early prognosis for those potentially aficted in the futureand perhaps still treatablebecomes nearly impossible. To halt disease progression would require a detailed understanding of the temporal sequence of events transpiring between the initial insult and the endpoint of massive neural cell death. The inability to either prevent or halt the disease process leaves those in the eld in the unfortunate position of at best being able to treat symptoms, but only once clinically identied. Currently, treatment is primarily palliative, though this may change in the future with the advent of stem cell or other future technological breakthroughs. However, due to signicant theoretical and economic factors, curing these diseases following clinical diagnosis may never be successful. Without a clear understanding of how various pathological processes arise and evolve in real time to cause neural death, especially in relation to outcomes at all levels of neural cell function, no therapeutic strategy can be successful. In order for us to advance beyond palliation, a multi-dimensional view of the disease processes is required. Such a view would encompass successive levels of neural organization, from behavior to cellular/biochemical function. Time comprises the fourth dimension of this analysis. The concept of a four dimensional analysis of neurological disease is further dened below. This article describes what is known about the temporal progression of human neurological diseases, AD, PD, ALS,

and ALS-parkinsonism dementia complex (ALS-PDC). We go on to relate this information to insights gained from a new animal model of ALS-PDC in which we can observe the evolution of neurodegeneration in each of the above dimensions.

2. Dening neurological disease in four dimensions To fully understand a progressive neurological disease, it must be observed across various neurological dimensions. In place of the three traditional physical dimensions of height, width, and breadth, our use of the word dimension is directed at various levels of neural organization. For the purpose of the present discussion, we dene these as: (i) biochemical processes which normally or abnormally occur in the various neural cell types, (ii) the normal or abnormal cellular morphologies associated with different neural cell types, and (iii) the normal or abnormal outcomes of the behavioral response. We acknowledge that there are additional sub-levels that can be discussed (for a more complete description of our concept of neural levels of organization see Ref. [1]) and that our choice of levels/ dimensions is arbitrary. Each of these levels is dynamically interactive with those above and below. For example, toxins that alter the biochemical makeup of a particular neural subtype (i.e. type I mitochondrial inhibitors acting on dopaminergic neurons) may kill the cell in question, thus disrupting the overall neural circuit in which that cell acted. In turn, disruptions of cellular interactions impact larger neural circuits and systems, ultimately altering the behavioral outcome. A full understanding of the degenerative process and the development of therapeutic strategies to block the neurodegeneration, requires an appreciation of the time course or timeline (see Fig. 1).

3. The Timeline concept The notion of a timeline is crucial for understanding the evolving cascade of pathological events in neurodegeneration. The simplest notion is that the timeline consists of a straightforward relationship between the presence of some toxicant molecule, gene or gene product and the number of dead or dying cells in affected regions of the central nervous system (CNS). For example, as the amount of a particular toxin increases, the cumulative number of dead cells should increase as well. In this example, the relationship is much like the well-known Michaelis Menton curve that describes enzyme-substrate reactions. Certainly, in neurological diseases the progression of neurodegeneration could follow mathematical functions such as this and might occur in cases where high concentrations of a specic and acutely toxic molecule were present. Another simple form of timeline might substitute time for dose, such that for a given dose of any toxic molecule, the cumulative number of

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Fig. 1. Schematic: neurological disease in four dimensions. The combined X ; Y ; and Z -axes represent an arbitrary region of CNS affected by a progressive neurodegenerative disorder of undened nature. The X -axis represents cell structure/function (neuron number/structure in particular regions of CNS), Y represents normal behavioral function, and Z represents biochemical processes in the CNS. Axis length corresponds to percent remaining function and has been arbitrarily set for this example. As the disease progresses, the biochemical malfunction leads to greater cell loss and ultimate decreased behavioral function. Behav: behavioral function. Cell struct: cell structure/function. Biochem: normal biochemical processes.

dead or dying cells would increase over time. An example of the latter function is the association binding kinetics of receptor ligand interactions. Although these simple examples may apply to neural cells in some constrained circumstances, e.g. in vitro for single cell types in cell culture preparations, they are unlikely to do so in vivo. The reasons why such simple relations are not likely to apply in vivo include the following: (i) in vivo, multiple cell types interact in any region of the nervous system; (ii) various cell types are differentially impacted by any toxin or gene; (iii) sub-acute toxin levels may initially damage rather than kill certain cells; and (iv) secondary and tertiary, etc. molecules released by one or multiple cell types can impact the health and survival of surrounding cells. In other words, the in vivo system is dynamic and reects an ongoing interplay of multiple cell types and numerous biochemical cascades over time. This consideration indicates that while any initial local toxic action may reect a simple toxin-induced destruction of particular cells, the longer term consequence will be a series of biochemical cascades of great complexity and involving numerous cell types. The latter circumstance is almost certainly the case in human chronic neurological disease states. In the present article, our use of the term timeline will refer generally to the more complex case.
Table 1 Comparison of ALS, PD, AD, and ALS-PDC Incidence per 100,000 ALS PD AD ALS-PDC 14 [3,82] 21 [3]; 19 in Estonia [84]; , 100200 on Als and Faroe Islands [85]; Greenland [86]; and Bulgaria [87] 39101 [89]; 401 [3] 19451960: 114 155 19951999: 27 [25]

4. Overview of age-related neurological diseases The age-related neurological diseases, including AD, PD, and ALS, are diagnosed only once signicant behavioral decits have been observed clinically. Alzheimers disease involves the death of neurons of various regions of the cerebral cortex and the hippocampus and results in the loss of cognitive functions such as memory and learning. In Parkinsons disease, portions of the nigral striatal system degenerate. Initial stages involve the loss of terminal projections of dopamine-containing neurons from the substantia nigra (SN). In turn, the neuron cell bodies in the SN die, impacting motor control and leading to tremor and gait disturbances. ALS primarily involves the loss of spinal and cortical motor neurons, leading to increasing paralysis and eventually death. Table 1 compares several aspects of ALS, AD, PD and ALS-PDC not further mentioned in this article. Each of these diseases appears to target relatively specic populations of neurons in the CNS whose loss leads to particular neurological symptoms at a behavioral level. The conventional perspective is that these are quite distinct diseases, arising from different etiologies, and expressing as unique behavioral and neuropathological outcomes. To some extent this view is justied due to differential primary

Male:female ratio 2:1 [83]; 1.6:1 [3] 2.1:1 [88]; 1.1:1 [3] 1:1.7 [90]; 1.1:1 (but ratio reverses after age 75 years) [3] 19451960: , 2:1 19951999: comparable rates [25]

Mean age of onset 59.3 [3] 61.9 [3] 71.9 [3] 19451960: 50 59 19951999: 65 69 [25]

ALS: amyotrophic lateral sclerosis, PD: Parkinsons disease, AD: Alzhiemers disease and ALS-PDC (ALS-parkinsonism dementia complex) are compared.

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symptoms and pathological outcomes of the diseases at a cellular and neural circuit level. However, recent research also reveals signicant commonalities and apparent boundary crossing amongst these disorders (see Refs. [2,3] for reviews). For example, patients suffering from AD may show tremor [4], a hallmark of PD. Similarly, PD and ALS patients may show losses of cognitive function [5 7], primarily an AD symptom. A recent article by Masliah et al. [8] has linked AD and PD as possibly having overlapping pathogenic pathways. Clinically, neurological gait abnormalities in elderly persons normally seen in PD patients have been shown to be a signicant predictor of the risk of developing later dementia [9]. Clinical observations have also dened a new disorder called ALS-plus, which is a form of ALS combined with dementia and/or parkinsonism features [10]. (For further examples of commonalities in theses diseases see Refs. [11 13]). At postmortem analysis, some of the hallmark features of the different disorders may cross conventional boundaries as well. For example, neurobrillary tangles (NFT), characteristic of AD, have now been identied in some cases of PD [14] and ALS [15]. Similarly, a-synuclein, a major component of Lewy bodies and Lewy neurites, the pathological hallmarks of PD, were originally isolated from amyloid plaques in AD patients [16]. Eisen and Calne [3] have suggested that AD, PD and ALS share more basic underlying features, e.g. generic protein misfolding alterations that differ in the types of proteins affected. For example, the protein alterations involving b-amyloid (AD), a-synuclein (PD) and heavy neurolaments (ALS) may suggest that an important step in neurodegeneration is altered cytoskeletal protein per se, rather than the particular protein involved. In each of the above diseases, by the time clinical diagnosis is achieved, major damage has been done to the specic region(s) of the nervous system most affected. Estimates of neuron loss in these areas vary, but may be extensive (e.g. 70% loss of functional spinal alpha motor neurons in ALS [17]; . 75% loss of neurons of the nucleus basalis of Meynert in AD [18]; 60% loss of the enzyme dopa-decarboxylase as a gauge of dopaminergic terminals in striatum in PD [19]). A recent study with AD patients using MRI volume measurements of medial temporal lobe structures (hippocampus and entorhinal cortex) showed a 16.6% decrease compared to controls [20]. Across the various diseases discussed here, neural compensation by surviving neurons may sustain the individual over long periods, at least until a nal threshold of functional neurons is lost. Clinical symptoms may become detectable only after severe damage beyond this threshold is done to the most affected neural subset(s).

5. Is there a neurological Rosetta Stone? A classical example of overlapping symptoms in a progressive neurological disease is the unusual Guamanian

disorder, ALS-PDC which rst gained serious attention in the 1950s. L.T. Kurland and various other investigators described in detail this disease complex, which could express as a conventional form of ALS (termed lytico or paralytico by the Chamorro population of Guam and Rota) or as a form of Alzheimers disease with strong parkinsonian features (locally termed bodig). A number of patients presented with a combination of features, often sequentially developed, with the ALS component usually appearing rst [21]. Kurland and other early investigators (for review, see Refs. [22,23]) thought the disorder remarkable in several key aspects. First, the overall incidence was vastly higher than for related disorders elsewhere (50 100 times more prevalent among the Chamorros of Guam than in the rest of the world, [24]), so much so that Kurland estimated that 25% of adult deaths on Guam/Rota were due to ALS-PDC. Second, the disease often struck those much younger than the average age of onset for similar disorders elsewhere (mean age onset 44 years [24] vs. 59 for ALS, 62 for PD and 72 for AD [3]). Finally, the overlapping features in many cases seemed to point to a common etiology, one that might shed light on all forms of age-related neurological disease. The view at that time was that ALS-PDC could serve as a type of neurological Rosetta Stone, the decipherment of which would unlock crucial clues to neurological disorders worldwide. Early studies of the environment and genetics of the Chamorro people gave hope that straightforward causal factors would be readily unearthed. For example, the Chamorro population was then relatively homogeneous in genetic background [25]. Additionally, Kurland and colleagues cited the relative lack of potential environmental toxins of human origin. In spite of this, detailed screening over many years failed to identify a genetic etiology (for a recent reference, see Ref. [25]). Largely for this reason, investigators rapidly focused on potential environmental toxins, screening hundreds of potential factors, including the ionic composition of ground water, native food products, industrial materials associated with military activity, and radiation. Most of these potential candidates were eliminated as being sole causal factors, although we note that controversy still remains about possible synergistic interactions between weak toxins and/or between weak toxins and possible genetic susceptibilities [25]. The primary clue to the cause of the disease was the historical record showing toxic effects of the seed of the cycad palm (Cycas micronesica K.D. Hill, previously referred to as Cycas circinalis), a traditional food often used as a primary foodstuff during times of famine. Cycad seeds were harvested, cut open to expose the starchy endosperm, sliced into chips, then washed for periods up to 10 days. The Chamorros had originally been introduced to cycad consumption by the Spanish who taught them to wash out acutely toxic factors (Steele, personal communication). It was noted that Captain Cooks sailors visiting the island in the late 1700s had consumed unwashed cycad and

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had become seriously ill, some showing acute neurological signs [26]. Cattle and other ruminants feeding on cycad in other locales died after exhibiting symptoms of neurological dysfunction [26]. As noted above, cycad had been consumed by the Chamorros as a dietary staple and occasional famine food, but the level of consumption rose to far greater levels during WWII due to harsh conditions during the Japanese occupation of the island. ALS-PDC incidence peaked within several years of the war and dramatically declined as cycad consumption lessened during the post-war years. Guamanians who adopted a more conventional Americanized diet showed declining incidences of ALS-PDC [19]. Notably, the Chamorro people of Saipan, genetically identical to the Chamorros of Guam, had not consumed cycad during most of the 20th Century and had no cases of ALS-PDC, with only one case of ALS and three cases of parkinsonism without dementia among the approximately 17,000 inhabitants. These rates were comparable to those in North America [21]. Thus, genetically identical populations showed two completely different outcomes, apparently based solely on exposure to one potential source of toxicity. Each of these factors led Kurland and others to conclude that cycad toxins were the key etiological factor in ALSPDC and this conclusion sparked a hunt for the principal toxin involved. The various cycad species are gymnosperms, appearing earlier in evolution than angiosperms, or owering plants [27]. While the latter use birds and mammals as a means to distribute seeds, cycads do not, and the leaves and seeds contain a number of compounds that are acutely toxic to mammals [28]. Some of these include the amino sugar cycasin and its active compound, the aglycone methyl azoxylmethanol (MAM), the latter being acutely toxic as well as having both carcinogenic and mutagenic properties [29]. Various amino acids able to activate subclasses of the ionotropic glutamate receptor family are also present, including b-N-oxalylamino-L alanine (BOAA) and b- N -methylamino- L -alanine (BMAA), agonists for AMPA and NMDA receptors, respectively. A number of other compounds have also been detected in cycad, some still not well characterized [30,31]. Investigators in the 1960s seized on the toxicity of MAM, using either cycad or the isolated toxin in a series of experiments designed to demonstrate both the neuronal effects as well as the mechanisms of action. Two facts gradually became apparent. First, animals exposed to cycasin/MAM or BMAA did not exhibit the full gamut of behavioral or pathological outcomes that resembled ALSPDC [32,33], although Spencer and colleagues did succeed in producing motor dysfunctions accompanied by loss of spinal motor neurons in monkeys with the latter toxin [34]. Second, both cycasin and MAM were signicantly eluted by the traditional washing procedure of the Chamorros [35]. Spencer et al. [36,37] did demonstrate that BMAA could induce pathological neurological outcomes in vitro [38] and in vivo [39]. A more chronic form of neuronal dysfunction

due to BOAA is lathyrism arising from the consumption of the chickling pea [40,41]. Recently, Cox and Sacks [42] suggested that the source of the ALS-PDC inducing toxin is indeed cycad, but that this toxicity is biomagnied by being stored in the bodies of fruit bats, the latter eaten by the Chamorros until the 1970s. Traditional washing of cycad chips as part of processing, however, removes most toxins [35], suggesting that various water soluble compounds are not primary factors responsible for ALS-PDC. This observation, however, does not discount the possibility of biomagnication in which the washing of the cycad seeds may become irrelevant. In spite of the ebb and ow of etiological hypotheses, the strongest epidemiological data for ALS-PDC still pointed to cycad consumption, a fact that led our group to reexamine the cycad hypothesis from the perspective that still unknown, water-insoluble cycad toxins might be causal to the disease. Some of the key conclusions of our studies are presented below and form the basis for our murine model of ALS-PDC.

6. A murine model of ALS-PDC We recently reexamined the cycad model of ALS-PDC using quantitative assay procedures combined with bioassays for neural activity and cell death [43]. These studies identied the most toxic types of molecules contained in washed cycad as a sterol glucoside whose actions in vitro included the excitotoxic release of glutamate and an abnormal increase in the activity of various protein kinases. We expanded our studies to include in vivo feeding of washed cycad seed our and employed a battery of motor, cognitive, and olfactory behavioral measures to determine the outcome of consumption. These studies demonstrated a temporal sequence of behavioral decits that correlated to neural cell death in appropriate regions of the CNS [7,43] (a summary is provided in Fig. 2). With regard to motor neuron disorders, cycad-fed mice showed signicant losses of the leg extension reex (Fig. 2C), pronounced gait disturbances (Fig. 2B), as well as losses of muscle strength and balance. MRI scans of the brains and spinal cords of control and cycad treated animals ex vivo showed decreased cross sectional areas of various regions of motor and somatosensory cortex, decreased volumes in hippocampus, substantia nigra/striatum, olfactory bulb and ventral horn of the spinal cord (Fig. 2D F). In addition, motor neuron number was decreased signicantly in ventral cord (Fig. 2F(i)). On sacrice, mice fed with cycad showed TUNEL and caspase-3 positive cells indicative of apoptosis in spinal cord, cortex, hippocampus, substantia nigra and olfactory bulb (Fig. 2G I). In addition to motor decits, observed regions of neural degeneration were consistent with observed cognitive and sensory decits. Both spatial learning (Morris water maze) and reference memory (radial arm maze)

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Fig. 2. Summary of data from the cycad mouse model. A C: behavioral decits displayed by cycad-fed mice (A) cycad-fed mice, (B) control mice. A: radial arm maze test of spatial memory. Reference memory (entrances into incorrect arms) and working memory (entrances to previously visited arms) errors by cycad-fed and control mice are shown. B: Paw Print testing for walking gait length. C: Leg extension reex test, a measure of motor neuron integrity. D F: Cell counts or MRI volume measurements in specic CNS areas of cycad-fed mice compared to controls. D: i. Cortical thickness measurement of several areas of cortex. V1: primary visual cortex. LEnt: lateral entorhinal cortex. M1: primary motor cortex. S1: primary somatosensory cortex. Pir: piriform cortex. ii. Hippocampus volumes of control and cycad-fed mice. E: i. Striatum volumes of control and cycad-fed mice. ii. Substantia nigra (SN) volumes of control and cycad-fed mice. F: i. Motor neuron counts from ventral horn of spinal cord from control and cycad-fed mice. ii. Ventral horn volumes of control and cycad-fed mice. GI: Histological display of cell pathology in CNS of cycad-fed mice. G: TUNEL labeling in cycad-fed mouse. i, ii: TUNEL labeling in cortex. iii. TUNEL labeling in Dentate gyrus. iiii 40 magnication. H: Caspase-3 labeling of cycad-fed mouse substantia nigra (SN). i. 10 magnication. ii. 40 magnication. I: Cresyl Violet staining of spinal cord motor neurons. i. Control, ii, iii. Cycad-fed mouse. JL: Examples of selected biochemical changes in CNS tissue from cycad-fed and control mice. J: Immunolabeling of GLT-1 in primary motor cortex,. i: Control, ii. Cycad-fed. K: Tyrosine hydroxalase (TH) labeling of striatum. i: Control, ii. Cycadfed. L: Immunolabeling of GLT-1 of spinal cord. i: Control, ii. Cycad-fed. J, L: scale bar 80 mm.All graphs show means ^ SEM, (* P , 0:05: A C, G I: Original data from [7]; D F: Original data from [81]; J, L: Original data from Ref. [44]; K: Original data from Ref. [45]).

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(Fig. 2A) tasks were degraded, with corresponding neurodegeneration seen in regions of cerebral cortex and hippocampus (Fig. 2D). In addition, the olfactory system showed a signicant loss of function accompanied by disrupted structures and cell loss in the olfactory glomeruli. In various regions, key molecules associated with neuronal degeneration were altered. These included an elevation of tau protein and various protein kinases, notably various PKC subtypes and CDK5. In addition, elements of the glutamatergic system were severely affected, most notably a dramatic decrease in levels of two variants of the GLT 1 glutamate transporter (EAAT2) (Fig. 2J and L) accompanied by a decrease in NMDA and AMPA receptor binding [44]. The apparent down-regulation in GLT and glutamate receptor subtypes was noted in regions of the CNS showing neural degeneration. Labeling for tyrosine hydroxylase revealed a signicant decrease of labeled terminals in striatum and of cell bodies in substantia nigra pars compacta of cycad fed mice (Fig. 2K) [45]. All of the features described above are consistent with features of ALS-PDC, as well as key aspects of AD, PD, and ALS. Of particular signicance is the observation that the alterations in behavior and CNS morphology are progressive in adult mice even after cycad exposure ends [7].

7. Does the ALS-PDC model satisfy standard criteria? A model is dened as any experimental preparation developed for the purpose of studying a condition in the same or different species [46]. When assessing any model it is critical to consider the explicit purpose of the model as this determines the criteria that must be satised to establish validity. In this regard, three components of validity must be satised: predictive, construct, and etiological. With regard to modeling human neurological disease, the latter assumes the greatest signicance and is a point on which most animal models fail. For example, b-amyloid mice display cognitive decits [8], but genetic induction of b-amyloid is not likely the cause of late onset AD in most patients [47,48]. Similarly, for ALS the SOD-1 mutant mouse is widely used, but mutant SOD in humans only accounts for 2% of all ALS cases (20% of familial ALS patients [49]). In the same vein, a-synuclein accumulation can be a feature of both sporadic and familial PD, but it has only been linked as the cause of the parkinsonism in a small number of families [50]. In contrast to these models, consumption of cycad in humans is the strongest epidemiological link to ALS-PDC and as described above similar feeding paradigms in mice produce neurological outcomes that mimic the disease in all essential features. Thus, the murine model of ALS-PDC meets the criterion of etiological validity. Construct validity is dened as the accuracy with which a test measures what it is intended to measure. Any animal model of a progressive neurological disease should provide

predictable time-dependent losses of neural cells in the same CNS regions affected in the human disease. The ALSPDC model satises this criterion since it demonstrates progressive neuronal loss in regions of CNS accompanied by appropriate behavioral dysfunction, both resembling measurable features of ALS-PDC. In addition, the phenomenology is robust in that it has been routinely observed in multiple batches of animals and displays the range of motor and cognitive outcomes that comprise the diverse expression of the various sub-disorders in ALS-PDC, i.e. ALS, PDC and the combined symptoms. The nal standardpredictive validityis dened as the ability of a test to predict a criterion that is of interest to the investigator [46]. In any neurodegenerative disease, the major criterion of interest is a progressive degeneration of specic neuronal subsets. For example, in Parkinsons disease, the major neuronal degeneration is in the substantia nigra and striatum; in ALS, degeneration of upper and/or lower motor neurons of the brain/spinal cord; AD is typied by degeneration of cortical neurons and cortical thinning along with the appearance of NFT and/or amyloid plaques. Our model of ALS-PDC meets this criterion as well: Mice fed with washed cycad our display progressive cognitive and motor behavioral decits as well as corresponding CNS pathologies. In addition, a clearly predictive feature of our model is the olfactory sensory decit and disruption of the morphology of olfactory glomeruli. These data predict similar decits in human AD, PD, ALS, and ALS-PDC, features which are now reported in Refs. [51 53].

8. Genetic epigenetic interactions in the cycad model Our results have clearly demonstrated that an exogenous neurotoxin contained in cycad can induce features of ALSPDC in a mouse model. These results lend strong support to the notion that many of the features of the human disease, as well as similar age-related neurodegenerative diseases, could have as their basis an environmental toxin to which various fractions of the population are exposed and susceptible. The notion that environmental toxins could be primary etiological factors has long been considered for various neurological diseases and a recent study on identical twins lends increasing support to the notion that environment, rather than a simple genetic factor, is crucial (see Ref. [54]). Nevertheless, familial forms of these diseases exist and in such cases there is abundant evidence for genetic causality of a limited scope. Animal models of genetic factors involved in human neurological diseases have provided much information about potential mechanisms leading to cell death. For example, transgenic mice over-expressing b-amyloid show cognitive losses and neuronal damage similar to AD and mutant superoxide dismutase (mSOD1) mice expressing a toxic gain of

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function mutation for SOD show a progressive degeneration of motor neurons. Given the above data, it is too simplistic to view any of the sporadic forms of neurological disease as solely the result of either genes or environment. In fact, a growing number of investigators have concluded that the intersection of genetic-epigenetic factors is involved. Fig. 3 illustrates this concept, showing the intersection of genetic susceptibility factors with environmental toxins, along with age as the critical third variable. This last factor is crucial, especially given that the diseases under discussion in this article all occur within specic age ranges, usually beginning in late middle age. We have begun to examine gene-environment interactions using our cycad mouse model due to the ability of this model system to reliably induce neurodegeneration in specic neural subsets. We have now combined cycad feeding with two genetic variants. First, APO E transgenic mice have been used to explore the interaction of cycad toxins with genetic susceptibility co-factors. APO E proteins are involved in cholesterol handling and the various allele variants have been implicated in AD, ALS, and ALS-PDC [55]. Second, transgenic mice over-expressing mutant human SOD (mSOD1) have been used to examine cycad interactions with a genetic causal factor. mSOD has been linked to some forms of familial ALS (FALS) [56]. APOE knockout (KO) mice fed cycad did not display signicant behavioral decits, in marked contrast to cycadfed wild-type (WT) APOE mice which showed signicant

decits across a range of functions [57]. Histology showed cell death in the substantia nigra, hippocampus, and striatum in cycad-fed WT mice, but not in cycad-fed APOE KO mice. Cycad-fed APOE KO mice also showed increased cholesterol levels without displaying cycadinduced damage to heart or liver [57]. (Experiments in which cycad will be fed to mice expressing specic APOE isoforms (E2, E3, and E4) are currently underway.) These data clearly demonstrate that certain genetic susceptibility factors can increase or decrease sensitivity to toxin exposure, reinforcing the notion that the interplay of genes and environment is likely part of the overall constellation of factors leading to neurodegenerative disease. With regard to this last point, additional complexity is certain to arise in future experiments as we examine the complete temporal sequence of events from initial toxic insult to cell death, especially for animals of different ages. mSOD mice fed cycad showed accelerated motor behavioral losses, but in a manner that was not simply the sum of cycad effects combined with those of the mutation [58]. For example, measurements of leg extension, an index of spinal motor neuron integrity, showed that mSOD mice fed cycad had a move rapid decline than either mSOD mice or cycad-fed wild type mice. Measurements of latency to fall on a rotarod test gave a very different picture with cycad-fed mSOD mice performing better than mSOD mice not exposed to cycad. These data are preliminary and await conrmation, but highlight again the notion that gene-environment interactions are likely to be complex.

9. Time line of neurodegenerative events in the cycad model As described above, our studies in cycad-fed mice have utilized a battery of behavioral, biochemical, and morphological measures to create a working time line for the emergence of behavioral and pathological outcomes beginning with the initial toxin insult and progressing through to animal death. The data clearly reveal that the various behaviors are impacted at different times following exposure to cycad toxins. We do not yet have corresponding biochemical and histological studies for each time point, but these data are now beginning to emerge from our ongoing studies. For example, we now know that the down-regulation of glutamate transporter subtypes is a relatively early event, likely occurring long before cell death [44]. Placed in context, the alterations in protein kinase C we have noted in previous experiments, are likely to occur even earlier and may possibly be involved in abnormal phosphorylation of the transporter leading to down-regulation and loss of function [44]. Current studies will extend this timeline from initial insult, through the period of overt expression of neurological outcomes, and the stages leading to neural cell death. Fig. 4 illustrates our progress to date and provides

Fig. 3. Potential synergies of causal and risk factors in sporadic neurological disease. Causal factors involved in such diseases may reect exposure to toxin(s) that can arise from the environment or as a result of individual biochemical processes. In the former case, the toxins may be synthetic or naturally occurring. The toxic factor is represented by the set on the left side of the diagram. The range of toxins effects run from left to right as low to high. Intersecting this is a set consisting of a genetic susceptibility factors that could arise due to genetic polymorphism in efciency of detoxication mechanisms (from right to left, expressed as high to low). Genes coding for transport proteins (e.g. APO E alleles) could also be involved. The intersection of these two sets describes the individuals who may be at risk of developing the neurological disorder. Note that the intersecting region can increase or decrease depending on strength of either variable. Intersecting these two sets is the variable of age with the risk factor increasing from young to old (bottom to top).

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Fig. 4. ALS-PDC mouse model timeline of various behavioral and pathological events. Events described are from data collected from cycadfed mice at the time point in which the event falls. Progressing Behaviour lists events occurring during the time from start of feeding to sacrice. The Sacrice column describes events that were found post mortem in CNS tissue. Predicted Progression describes hypothetical progression of the cycad-induced outcomes. Arrows indicate predicted continuations ( ! ) or predicted originations ( ) of the events described. " : increasing levels/amounts. # : decreasing levels/amounts. Abbreviations: MNs: ventral horn spinal cord motor neurons; NFT: neurobrillary tangles.

a current interpretation of where the various behavioral and pathological events may be found along the timeline. Once the model timeline is completed, we will be in a position to attempt therapeutic interventions in our treated animals at each stage of the disease process to prevent further deterioration. Even if successful, however, ultimately we will need to transpose these data to the human population in order that such treatments can be directed to the right target at the precise time to have the greatest impact. The template matching between data from animal models and human disease states, especially preclinical states, remains the greatest single challenge for treating neurological disease patients.

10. Template matching to human neurological disease What is actually known about human neurological disease progression? A search of the literature reveals that the rates of decline for the various neurological diseases vary
Table 2 Disease progression of human and animal modeled neurodegenerative disorders AD Human (post-diagnosis progression) Disease progression roughly constant with time [65] PD

quantitatively for each disease post clinical diagnosis, but that the decline function is usually linear. For example in ALS, progression is linear for the decline of motor neurons [59] as is the risk of death [60], but these measures depend on the time of diagnosis [61]. For PD, there is a variable course of progression of different PD symptoms depending on age of onset, but these are always linear [6264]. For AD, disease progression measured by cognitive decline varies depending on sex and age of behavioral onset [65]. Unfortunately, lling in more details is unlikely to offer much in the way of treatment options. Of greater concern, there seems to be very little data about pre-clinical stages of these diseases, which may show different rates of decline compared to postdiagnosis. For example, loss of function (strength and functional activities) is linear, but the loss of motor neurons may be exponential with an initial rapid fall that precedes diagnosis followed by a more linear motor neuron loss as the disease progresses [66]. However, it is during the pre-clinical stage that the hope for effective prophylaxis or early treatment exists. This pre-clinical gap is where animal models can make the greatest contribution, for by extending the timeline backwards to disease-initiating factors they allow us to focus attention on the earliest potentially treatable stages of each disease. Although results are scattered and incomplete, some attempts have been made in this regard. For example, in the amyloid b model of AD, researchers have shown an exponential increase in amyloid b deposits [67]. Similarly, mSOD mice used to model ALS show exponentially increasing microglial activation from 0 to 120 days [68]. Some examples from the literature are summarized in Table 2. In regard to our own model, cycadfed mice show exponential decays in motor and cognitive functions, interspersed with what appear to be temporary surges in behavioral compensation (see Refs. [7,69]). In Fig. 5 we merge the two data sets, including the ALSPDC model system data with those from studies of human age-dependent neurological disease. Note that the former

ALS Linear decline of motor neurons [59]

Variable course of progression of different PD symptoms depending on age of onset, but always linear [62] Constant risk of death (Fig. 3a) [63] Dopaminergic degeneration in PD appears to slow down during course of the disease [64] Exponentially decreasing risk in chemically induced model of PD (Fig. 3D) [63]

Constant risk of death, but depending on time of diagnosis [60]

Mouse models (entire disease progression)

Exponential increase of Amyloid B deposits in PS1 APP transgenic AD mouse [67]

Exponentially increasing microglial activation from 0120 days (Fig. 3) in mSOD1 [68]

Disease progression rates are compared for Alzheimers disease (AD), Parkinsons disease (PD) and amyotrophic lateral sclerosis (ALS). Human disease rates are determined from beginning of behavioral symptoms, while animal models measure progression from onset to death.

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Fig. 5. Predicted timeline of human neurological diseases. Symptoms and pathological outcomes from Alzheimers disease, Parkinsons disease and ALS are described in a timeline of events. Preclinical symptoms describe events that are thought to occur prior to overt behaviour symptoms seen at clinical diagnosis. Features at death describe postmortem pathological ndings and end-state behavioral decits. Arrows indicate predicted continuations ( ! ) or predicted origins ( ) of events described. " : increasing levels/amounts. # : decreasing levels/amounts.

starts from the initial insult and moves forward in time to the end state condition, while the latter attempts to reverse engineer the earliest stages from the end state. Neither data set alone is sufcient, but the hope is that the future success of our model will allow us to ll in the enormous gaps that now exist concerning the timeline of pathological events in human neurological diseases. As discussed above, the problem is that we have no timeline to speak of for tracing the course of age-dependent human neurological diseases. How, for example, can we know who is pre-symptomatic for any of these diseases, especially any of the sporadic varieties? In principle, various brain scans or biomarkers could be widely employed, but the rst is not economically feasible for the population at large; the second would depend on the identication of crucial molecules, most of these still unknown. The necessary crossover and template matching from an animal model to human neurological disease states is a twofold process. First, we have to be able to place what we know from our model system into a clear description of events in all four dimensions. Second, armed with this information, we can then attempt template matching to pre- and post-clinical human patients. This procedure may be a time consuming one, yet offers, in our view, the only realistic possibility for prevention and early treatment of human neurological disease. We discuss a potential strategy in more detail below. 11. The mouse model of ALS-PDC: implications for prophylaxis and for halting disease progression Our work has demonstrated the following: First, we have been able to show that a toxin contained in cycad is able to kill neurons. Mice fed with washed cycad develop the same behavioral and pathological outcomes as ALS-PDC victims. Second, we have been able to show clear interactions of cycad toxicity with various susceptibility (APO E) or causal (mSOD) gene abnormalities. Third, we have begun the long process of dening the various stages leading from toxic insult to neurodegeneration.

Each of these outcomes has potentially enormous implications for detecting and treating neurological disease. The identication of potential toxins should now spark a search for these molecules and molecules sharing their mode of action in the human environment. The isolation and partial characterization of the toxicity of various sterol glucosides as the toxins giving rise to ALS-PDC may suggest that an important future goal is the identication of the sources of these toxins. Tracing such toxins would naturally include a search for them in food products other than in cycad. For example, soybeans may contain sterol glucoside levels greater than those of cycad (Soybean our: 214.9 mg/g [70]; Cycad: 11.5 84.5 mg/g [28,43]), although whether processed soy does so as well is still an unanswered question. In addition, sterol glucosides may be present in other sources, i.e. a data base search reveals that sterol glucosides are found in tobacco and survive in tobacco smoke [71,72]. A recent report also shows that Helicobacter pylori bacteria make a similar sterol glucoside, specically a cholesterol glucoside, [73,74] (note that Khabazian et al. [43], found cholesterol glucoside to be extremely neurotoxic in vitro). H. pylori infections are associated with increased risk for PD [75] and this potential link to PD, and the possible relation to other neurological disorders, should be examined in greater detail. Sterol glucosides are only one type of molecule able to induce the types of neurodegeneration seen in our model and in human neurological disease. Further, the sterol glucosides identied by us may contribute only a small fraction of total disease cases. From this, two possibilities arise: rst, structurally dissimilar molecules may have similar mechanisms of action. Second, sterol glucosides may interact synergistically with molecules having very different mechanisms of action on neurons. In the latter case, we consider the possibility of such interactions between excitotoxins and oxidative stress (see Ref. [76]). Overall, the identication of potential neurotoxins may serve as a rst means of prophylaxis since if we can detect such molecules in the environment, we may be able to avoid them. Of equal importance, the notion that certain genetic susceptibilities may be crucial, ties in with the identication of putative toxins, and can be explored in several ways. First, one could screen for the obvious gene candidates: SOD, APO E, etc. A more sophisticated search would look for genes involved in some process with which the putative toxin could interact. For example, our identication of sterol glucosides, including cholesterol glucoside, as potential neurotoxins in neurological disease is directly relevant to the notion that APO E allele variants can contribute to the expression of such diseases. Similarly, genes coding for the synthesis or modication of other sterols, e.g. the various CYP genes [77,78], for example, may be important. In addition, genes that control enzymes involved in the synthesis or degradation of sterol glucosides could be crucial [79,80]. The identication of human genetic proles,

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especially as they relate to environmental toxins, may serve to identify those persons at future risk of developing or expressing the disease. Prophylaxis may be achieved by identication of the putative toxin or by the identication of those whose genetic susceptibility makes them vulnerable to that toxin. As valuable as prophylaxis would be to those not yet affected, what does any of this imply for those at early and middle stages of exposure and presumed emerging neural damage? How are these individuals to be identied in order that targeted therapeutics halt the biochemical cascades from culminating in neurodegeneration and the clinical manifestation of disease? The answer to this must arise from a thorough understanding of the stages of the disease process and, as discussed, this can only occur via a comprehensive animal model that combines behavioral, biochemical, and histological analyses over an extended time period. In addition, an understanding of the full fourdimensional aspects of the disease process will allow targeted therapeutics to be applied to halt degenerative cascades. For example, early glutamate transporter abnormalities [44] created by excessive kinase activity might be reversed by either a selective block of that kinase or an induced up-regulation of the transporter. The timely application of therapeutic agents, at the right place and time, will be key components of a successful treatment paradigm. The challenge, however, is to extrapolate the animal fourdimensional data to the pre-clinical human population. We envision a process something like the following: The mouse timeline provides not only sequenced events leading to neurodegeneration, but also comparative behavioral data and, perhaps, the identication of various biomarkers for early stages of the disease process. Working backwards from patients currently diagnosed with the various diseases, we can attempt strong correlations to earlier pre-clinical behavioral changes. These, in turn, may lead to attempts to identify such behavioral outcomes in pre-clinical populations. A good example of this process would be the use of the apparent early olfactory disturbance in PD [51,52] to place these individuals into the correct position in the timeline. Behavioral indices of altered neural function might then trigger the search for identied biomarkers, the latter symptomatic of unique changes within the nervous system at particular time points. MRI or other imaging methods could then be employed to conrm changes to neural morphology. As above, template matching between human patients and animal models may allow a blockade of the neurodegenerative cascades at an early stage of the disease progression, when most behavioural function is still intact.

ALS. The exceptional features of ALS-PDC may suggest that AD, PD, and ALS share some common features and may arise in part due to common etiologies. The ALS-PDC behavioral and neuropathological outcomes can be reproduced in an animal model based on the consumption of toxins contained in cycad seeds. The data obtained by this model include the possible identication of the putative neurotoxin as well as a preliminary understanding of the temporal progression of neurodegeneration following exposure. In addition, we have begun to explore aspects of gene environment interactions as determinants of neurodegeneration. The insights gained by use of this model may allow for future prophylaxis and treatment of human neurological diseases. Acknowledgements This work was supported by grants from the ALS Association, Scottish Rite Charitable Foundation of Canada, Natural Science and Engineering Research Council of Canada, and the US Army Medical Research and Materiel Command (#DAMD17-02-1-0678) (to CAS). The authors thank Drs S. Blackband and S. Grant of the University of Florida, USA and D. Pow of the University of Queensland, Australia for collaborations on MRI imaging and glutamate transporter labeling, respectively. The authors also thank H. Bavinton, C Melder, and M. Wong for comments on the manuscript. Cycad seeds were provided by our colleagues on Guam, Drs U. Craig and T. Marler to whom we are very grateful. This review acknowledges the unique contributions of the late Dr L.T. Kurland. References
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12. Conclusion ALS-PDC is a unique disease that displays aspects of the major progressive neurodegenerative diseases, AD, PD and

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