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Review

Analysis of neurological disease in four dimensions: insightfrom ALS-PDC epidemiology and animal models

C.A. Shaw a,b,c,d, *,1 , J.M.B. Wilson a,1

aProgram in Neuroscience, University of British Columbia, Vancouver, BC, Canadab Department of Ophthalmology, University of British Columbia, Vancouver, BC, Canada

c

Department of Physiology, University of British Columbia, Vancouver, BC, Canadad Department of Experimental Medicine, University of British Columbia, Vancouver, BC, Canada

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 undenedand poorly understood. We have developed an animal model of amyotrophic lateral sclerosis-parkinsonism dementia complex which mimicsall the essential features of the disease with the initial neurological insult arising from neurotoxins contained in washed cycad seeds. Animalsfed washed cycad develop decits in motor, cognitive, and sensory behaviors that correlate with the loss of neurons in specic regions of thecentral nervous system. The ability to recreate the disease by exposure to cycad allows us to extend the model in multiple dimensions byanalyzing behavioral, cellular, and biochemical changes over time. In addition, the ability to induce toxin-based neurodegeneration allows usto probe the interactions between genetic and epigenetic factors. Our results show that the impact of both genetic causal and susceptibilityfactors 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

Contents1. Introduction: the fundamental problems in neurological disease research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0002. Dening neurological disease in four dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0003. The Timeline concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0004. Overview of age-related neurological diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0005. Is there a neurological Rosetta Stone?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0006. A murine model of ALS-PDC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0007. Does the ALS-PDC model satisfy standard criteria? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0008. Genetic epigenetic interactions in the cycad model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0009. Time line of neurodegenerative events in the cycad model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00010. Template matching to human neurological disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00011. The mouse model of ALS-PDC: implications for prophylaxis and for halting disease progression . . . . . . . . . . . . . . 00012. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

1. Introduction: the fundamental problemsin neurological disease research

Of all the diseases that humans have sought to understandand thereby control, those involving the nervous system have

0149-7634/$ - see front matter q 2003 Published by Elsevier Ltd.doi:10.1016/j.neubiorev.2003.08.001

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

1 Equal co-authors.

* Corresponding author. Address: Research Pavilion, VGH, Rm 386, 828West 10th Ave., Vancouver, BC, Canada V6T 1Z3. Tel.: 1-604-875-

4111x68375; fax: 1-604-875-4376.E-mail address: [email protected] (C.A. Shaw).

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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) andamyotrophic lateral sclerosis (ALS)) are the most commonand the most difcult to track in time given that they may beinitiated early in life yetnormally express in late middleto oldage.Forthese diseases, there are neitherdenitive hypothesesnor unambiguous experimental data showing what the causalfactors are likely to be. In particular, we are still uncertainwhether these diseases have genetic and/or environmentalorigins and how factors associated with either can inuenceneural cell survival. It is also unclear how pathologicalprocesses evolve from the initial insult(s) through to celldeath, or what the functional relationships are between neuralpathology and behavioral outcome at any stage of the disease

process. Postmortem studies have identied numerousmolecules that appear to be altered in amount or function,but the larger context or interrelationships between suchmolecules, and the temporal sequence of changes in theaffected cells or neural systems are largely unknown. Detailsabout rates of progression from clinical diagnosis to death areknown, but minimal details are available about early pre-clinical events before classical symptoms are observed.

Therapeutic intervention has proven to be difcult due tothe lack of a clearly established link to any of the putativecausal factors for neurological disease. The prevention of disease initiation becomes challenging without knowing inadvance which individuals are susceptible and for what

reasons. Similarly, without understanding or being able toidentify pre-clinical symptoms, early prognosis for thosepotentially aficted in the futureand perhaps stilltreatablebecomes nearly impossible. To halt diseaseprogression would require a detailed understanding of thetemporal sequence of events transpiring between the initialinsult and the endpoint of massive neural cell death. Theinability to either prevent or halt the disease process leavesthose in the eld in the unfortunate position of at best beingable to treat symptoms, but only once clinically identied.Currently, treatment is primarily palliative, though this maychange in the future with the advent of stem cell or otherfuture technological breakthroughs. However, due tosignicant theoretical and economic factors, curing thesediseases following clinical diagnosis may never be success-ful. Without a clear understanding of how variouspathological processes arise and evolve in real time tocause neural death, especially in relation to outcomes at alllevels of neural cell function, no therapeutic strategy can besuccessful. In order for us to advance beyond palliation, amulti-dimensional view of the disease processes isrequired. Such a view would encompass successive levels of neural organization, from behavior to cellular/biochemicalfunction. Time comprises the fourth dimension of thisanalysis. The concept of a four dimensional analysis of neurological disease is further dened below.

This article describes what is known about the temporalprogression of human neurological diseases, AD, PD, ALS,

and ALS-parkinsonism dementia complex (ALS-PDC). Wego on to relate this information to insights gained from anew animal model of ALS-PDC in which we can observethe evolution of neurodegeneration in each of the abovedimensions.

2. Dening neurological disease in four dimensions

To fully understand a progressive neurological disease, itmust be observed across various neurological dimensions.In place of the three traditional physical dimensions of height, width, and breadth, our use of the word dimensionis directed at various levels of neural organization. For thepurpose of the present discussion, we dene these as: (i)

biochemical processes which normally or abnormally occurin the various neural cell types, (ii) the normal or abnormalcellular morphologies associated with different neural celltypes, and (iii) the normal or abnormal outcomes of thebehavioral response. We acknowledge that there areadditional sub-levels that can be discussed (for a morecomplete 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 dynamicallyinteractive with those above and below. For example,toxins that alter the biochemical makeup of a particularneural subtype (i.e. type I mitochondrial inhibitors acting ondopaminergic 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 largerneural circuits and systems, ultimately altering the beha-vioral outcome. A full understanding of the degenerativeprocess and the development of therapeutic strategies toblock the neurodegeneration, requires an appreciation of thetime course or timeline (see Fig. 1).

3. The Timeline concept

The notion of a timeline is crucial for understanding theevolving cascade of pathological events in neurodegenera-tion. The simplest notion is that the timeline consists of astraightforward relationship between the presence of sometoxicant molecule, gene or gene product and the number of dead or dying cells in affected regions of the central nervoussystem (CNS). For example, as the amount of a particulartoxin increases, the cumulative number of dead cells shouldincrease as well. In this example, the relationship is muchlike the well-known Michaelis Menton curve thatdescribes enzyme-substrate reactions. Certainly, in neuro-logical diseases the progression of neurodegeneration could follow mathematical functions such as this and might occurin cases where high concentrations of a specic and acutelytoxic molecule were present. Another simple form of timeline might substitute time for dose, such that for agiven dose of any toxic molecule, the cumulative number of

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dead or dying cells would increase over time. An example of the latter function is the association binding kinetics of receptorligand interactions.

Although these simple examples may apply to neuralcells in some constrained circumstances, e.g. in vitro forsingle cell types in cell culture preparations, they areunlikely to do so in vivo. The reasons why such simplerelations are not likely to apply in vivo include thefollowing: (i) in vivo, multiple cell types interact in anyregion of the nervous system; (ii) various cell types aredifferentially impacted by any toxin or gene; (iii) sub-acutetoxin levels may initially damage rather than kill certaincells; and (iv) secondary and tertiary, etc. moleculesreleased by one or multiple cell types can impact the healthand survival of surrounding cells. In other words, the in vivosystem is dynamic and reects an ongoing interplay of multiple cell types and numerous biochemical cascades overtime. This consideration indicates that while any initial localtoxic action may reect a simple toxin-induced destructionof particular cells, the longer term consequence will be aseries of biochemical cascades of great complexity andinvolving numerous cell types. The latter circumstance isalmost certainly the case in human chronic neurological

disease states. In the present article, our use of the termtimeline will refer generally to the more complex case.

4. Overview of age-related neurological diseases

The age-related neurological diseases, including AD,PD, and ALS, are diagnosed only once signicantbehavioral decits have been observed clinically. Alzhei-mers disease involves the death of neurons of variousregions of the cerebral cortex and the hippocampus andresults in the loss of cognitive functions such as memoryand learning. In Parkinsons disease, portions of thenigralstriatal system degenerate. Initial stages involvethe loss of terminal projections of dopamine-containingneurons from the substantia nigra (SN). In turn, theneuron cell bodies in the SN die, impacting motorcontrol and leading to tremor and gait disturbances. ALSprimarily involves the loss of spinal and cortical motorneurons, leading to increasing paralysis and eventuallydeath. Table 1 compares several aspects of ALS, AD, PDand ALS-PDC not further mentioned in this article.

Each of these diseases appears to target relatively specicpopulations of neurons in the CNS whose loss leads toparticular neurological symptoms at a behavioral level. Theconventional perspective is that these are quite distinctdiseases, arising from different etiologies, and expressing as

unique behavioraland neuropathological outcomes.To someextent this view is justied due to differential primary

Fig. 1. Schematic: neurological disease in four dimensions. The combined X ; Y ; and Z -axes represent an arbitrary region of CNS affected by a progressiveneurodegenerative 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 beenarbitrarily 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.

Table 1Comparison of ALS, PD, AD, and ALS-PDC

Incidence per 100,000 Male:female ratio Mean age of onset

ALS 14 [3,82] 2:1 [83] ; 1.6:1 [3] 59.3 [3]PD 21 [3]; 19 in Estonia [84]; , 100200 on Als and

Faroe Islands [85] ; Greenland [86] ; and Bulgaria [87]2.1:1 [88] ; 1.1:1 [3] 61.9 [3]

AD 39101 [89]; 401 [3] 1:1.7 [90] ; 1.1:1 (but ratio reversesafter age 75 years) [3]

71.9 [3]

ALS-PDC 19451960: 114155 19451960: , 2:1 19451960: 505919951999: 27 [25] 19951999: comparable rates [25] 19951999: 6569 [25]

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

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symptoms and pathological outcomes of the diseases at acellular and neural circuit level. However, recent researchalso reveals signicant commonalities and apparent bound-ary crossing amongst these disorders (see Refs. [2,3] forreviews). Forexample,patientssufferingfrom AD mayshowtremor [4], a hallmark of PD. Similarly, PD and ALS patientsmay show losses of cognitive function [57], primarily anAD symptom. A recentarticle by Masliah et al. [8] has linkedAD and PD as possibly having overlapping pathogenicpathways. Clinically, neurological gait abnormalities inelderly persons normally seen in PD patients have beenshown to be a signicant predictor of the risk of developinglater dementia [9]. Clinical observations have also dened anew disorder called ALS-plus, which is a form of ALScombined with dementia and/or parkinsonism features [10] .

(For further examples of commonalities in theses diseasessee Refs. [1113] ).At postmortem analysis, some of the hallmark features of

the different disorders may cross conventional boundaries aswell. For example, neurobrillary tangles (NFT), charac-teristic of AD, have now been identied in some cases of PD[14] and ALS [15] . Similarly, a -synuclein, a majorcomponent of Lewy bodies and Lewy neurites, thepathological hallmarks of PD, were originally isolatedfrom amyloid plaques in AD patients [16] .

Eisen and Calne [3] have suggested that AD, PD andALS share more basic underlying features, e.g. genericprotein misfolding alterations that differ in the types of

proteins affected. For example, the protein alterationsinvolving b -amyloid (AD), a -synuclein (PD) and heavyneurolaments (ALS) may suggest that an important step inneurodegeneration is altered cytoskeletal protein per se,rather than the particular protein involved.

Ineach of theabove diseases, by thetimeclinicaldiagnosisis achieved, major damage has been done to the specicregion(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 thenucleus basalis of Meynertin AD [18] ; 60% loss of the enzyme dopa-decarboxylase as agauge of dopaminergic terminals in striatum in PD [19]). Arecent study with AD patients using MRI volume measure-ments of medial temporal lobe structures (hippocampus andentorhinal cortex) showed a 16.6% decrease compared tocontrols [20] . Across the various diseases discussed here,neural compensation by surviving neurons may sustain theindividual over long periods, at least until a nal threshold of functional neurons is lost. Clinical symptoms may becomedetectable only after severe damage beyond this threshold isdone to the most affected neural subset(s).

5. Is there a neurological Rosetta Stone?

A classical example of overlapping symptoms in aprogressive neurological disease is the unusual Guamanian

disorder, ALS-PDC which rst gained serious attention inthe 1950s. L.T. Kurland and various other investigatorsdescribed in detail this disease complex, which couldexpress as a conventional form of ALS (termed lytico orparalytico by the Chamorro population of Guam and Rota)or as a form of Alzheimers disease with strong parkinso-nian features (locally termed bodig). A number of patientspresented with a combination of features, often sequentiallydeveloped, with the ALS component usually appearing rst[21]. Kurland and other early investigators (for review, seeRefs. [22,23] ) thought the disorder remarkable in severalkey aspects. First, the overall incidence was vastly higherthan for related disorders elsewhere (50100 times moreprevalent 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 thanthe average age of onset for similar disorders elsewhere(mean age onset 44 years [24] vs. 59 for ALS, 62 for PD and72 for AD [3]). Finally, the overlapping features in manycases seemed to point to a common etiology, one that mightshed light on all forms of age-related neurological disease.The view at that time was that ALS-PDC could serve as atype of neurological Rosetta Stone, the decipherment of which would unlock crucial clues to neurological disordersworldwide.

Early studies of the environment and genetics of theChamorro people gave hope that straightforward causal

factors would be readily unearthed. For example, theChamorro population was then relatively homogeneous ingenetic background [25]. Additionally, Kurland and col-leagues cited the relative lack of potential environmentaltoxins of human origin. In spite of this, detailed screeningover many years failed to identify a genetic etiology (for arecent reference, see Ref. [25] ). Largely for this reason,investigators rapidly focused on potential environmentaltoxins, screening hundreds of potential factors, includingthe ionic composition of ground water, native food products,industrial materials associated with military activity, andradiation. Most of these potential candidates were elimi-nated as being sole causal factors, although we note thatcontroversy still remains about possible synergistic inter-actions between weak toxins and/or between weak toxinsand possible genetic susceptibilities [25].

The primary clue to the cause of the disease was thehistorical record showing toxic effects of the seed of thecycad palm ( Cycas micronesica K.D. Hill, previouslyreferred to as Cycas circinalis ), a traditional food oftenused as a primary foodstuff during times of famine. Cycadseeds were harvested, cut open to expose the starchyendosperm, sliced into chips, then washed for periods upto 10 days. The Chamorros had originally been introducedto cycad consumption by the Spanish who taught them towash out acutely toxic factors (Steele, personal communi-cation). It was noted that Captain Cooks sailors visiting theisland in the late 1700s had consumed unwashed cycad and


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had become seriously ill, some showing acute neurologicalsigns [26]. Cattle and other ruminants feeding on cycad inother locales died after exhibiting symptoms of neurologicaldysfunction [26] . As noted above, cycad had beenconsumed by the Chamorros as a dietary staple andoccasional famine food, but the level of consumption roseto far greater levels during WWII due to harsh conditionsduring the Japanese occupation of the island. ALS-PDCincidence peaked within several years of the war anddramatically declined as cycad consumption lessenedduring the post-war years. Guamanians who adopted amore conventional Americanized diet showed decliningincidences of ALS-PDC [19] . Notably, the Chamorropeople of Saipan, genetically identical to the Chamorrosof Guam, had not consumed cycad during most of the 20th

Century and had no cases of ALS-PDC, with only one caseof ALS and three cases of parkinsonism without dementiaamong the approximately 17,000 inhabitants. These rateswere comparable to those in North America [21] . Thus,genetically identical populations showed two completelydifferent outcomes, apparently based solely on exposure toone potential source of toxicity.

Each of these factors led Kurland and others to concludethat cycad toxins were the key etiological factor in ALS-PDC and this conclusion sparked a hunt for the principaltoxin involved. The various cycad species are gymnos-perms, appearing earlier in evolution than angiosperms, orowering 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 compoundsthat are acutely toxic to mammals [28]. Some of theseinclude the amino sugar cycasin and its active compound,the aglycone methyl azoxylmethanol (MAM), the latterbeing acutely toxic as well as having both carcinogenic andmutagenic properties [29] . Various amino acids able toactivate subclasses of the ionotropic glutamate receptorfamily are also present, including b - N -oxalylamino- L-a lanine (BOAA) and b - N -methylamino- L -alanine(BMAA), agonists for AMPA and NMDA receptors,respectively. A number of other compounds have alsobeen 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 neuronaleffects as well as the mechanisms of action. Two factsgradually became apparent. First, animals exposed tocycasin/MAM or BMAA did not exhibit the full gamut of behavioral or pathological outcomes that resembled ALS-PDC [32,33] , although Spencer and colleagues did succeedin 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 bythe traditional washing procedure of the Chamorros [35].Spencer et al. [36,37] did demonstrate that BMAA couldinduce pathological neurological outcomes in vitro [38] andin 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 isindeed cycad, but that this toxicity is biomagnied by beingstored in the bodies of fruit bats, the latter eaten by theChamorros until the 1970s.

Traditional washing of cycad chips as part of processing,however, removes most toxins [35], suggesting that variouswater soluble compounds are not primary factors respon-sible for ALS-PDC. This observation, however, does notdiscount the possibility of biomagnication in which thewashing of the cycad seeds may become irrelevant.

In spite of the ebb and ow of etiological hypotheses, thestrongest epidemiological data for ALS-PDC still pointed tocycad consumption, a fact that led our group to reexamine

the cycad hypothesis from the perspective that stillunknown, water-insoluble cycad toxins might be causal tothe disease. Some of the key conclusions of our studies arepresented 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-PDCusingquantitative assay procedurescombinedwithbioassaysfor neural activity and cell death [43] . These studies

identied the most toxic types of molecules contained inwashed cycad as a sterol glucoside whose actions in vitroincluded the excitotoxic release of glutamate and anabnormal 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 determinethe outcome of consumption. These studies demonstrated atemporal sequence of behavioral decits that correlated toneural cell death in appropriate regions of the CNS [7,43] (asummary is provided in Fig. 2). With regard to motor neurondisorders, cycad-fedmiceshowed signicant losses of the legextension 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 cycadtreated animals ex vivo showed decreased cross sectionalareas of various regions of motor and somatosensory cortex,decreased volumes in hippocampus, substantia nigra/stria-tum, olfactory bulb and ventral horn of the spinal cord(Fig. 2DF ). In addition, motor neuron number wasdecreased signicantly in ventral cord ( Fig. 2F(i) ). Onsacrice,mice fedwith cycad showedTUNEL andcaspase-3positive cells indicative of apoptosis in spinal cord, cortex,hippocampus, substantia nigra and olfactory bulb(Fig. 2GI ). In addition to motor decits, observed regionsof neural degeneration were consistent with observedcognitive and sensory decits. Both spatial learning (Morriswater maze) and reference memory (radial arm maze)


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Fig. 2. Summary of data from the cycad mouse model. AC: 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 neuronintegrity. D F: Cell counts or MRI volume measurements in specic CNS areas of cycad-fed mice compared to controls. D: i. Cortical thicknessmeasurement of several areas of cortex. V1: primary visual cortex. LEnt: lateral entorhinal cortex. M1: primary motor cortex. S1: primarysomatosensory cortex. Pir: piriform cortex. ii. Hippocampus volumes of control and cycad-fed mice. E: i. Striatum volumes of control and cycad-fedmice. ii. Substantia nigra (SN) volumes of control and cycad-fed mice. F: i. Motor neuron counts from ventral horn of spinal cord from control andcycad-fed mice. ii. Ventral horn volumes of control and cycad-fed mice. G I: 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-3labeling of cycad-fed mouse substantia nigra (SN). i. 10 magnication. ii. 40 magnication. I: Cresyl Violet staining of spinal cord motorneurons. 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. Cycad-fed. 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 : AC,GI: Original data from [7]; DF: 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 neuro-degeneration seen in regions of cerebral cortex andhippocampus ( Fig. 2D). In addition, the olfactory systemshowed a signicant loss of function accompanied bydisrupted structures and cell loss in the olfactory glomeruli.

In various regions, key molecules associated withneuronal degeneration were altered. These included anelevation of tau protein and various protein kinases, notablyvarious PKC subtypes and CDK5. In addition, elements of the glutamatergic system were severely affected, mostnotably a dramatic decrease in levels of two variants of theGLT1 glutamate transporter (EAAT2) ( Fig. 2J and L )accompanied by a decrease in NMDA and AMPA receptorbinding [44]. The apparent down-regulation in GLT andglutamate receptor subtypes was noted in regions of the

CNS showing neural degeneration. Labeling for tyrosinehydroxylase revealed a signicant decrease of labeledterminals in striatum and of cell bodies in substantia nigrapars compacta of cycad fed mice ( Fig. 2K ) [45].

All of the features described above are consistent withfeatures of ALS-PDC, as well as key aspects of AD, PD, andALS. Of particular signicance is the observation that thealterations in behavior and CNS morphology are progress-ive 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 preparationdeveloped for the purpose of studying a condition in thesame or different species [46] . When assessing any model itis critical to consider the explicit purpose of the model asthis determines the criteria that must be satised to establishvalidity. In this regard, three components of validity must besatised: predictive, construct, and etiological. With regardto modeling human neurological disease, the latter assumesthe greatest signicance and is a point on which most animalmodels fail. For example, b -amyloid mice display cognitivedecits [8], but genetic induction of b -amyloid is not likelythe 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 ALScases (20% of familial ALS patients [49]). In the same vein,a -synuclein accumulation can be a feature of both sporadicand familial PD, but it has only been linked as the cause of the parkinsonism in a small number of families [50]. Incontrast to these models, consumption of cycad in humans isthe strongest epidemiological link to ALS-PDC and asdescribed above similar feeding paradigms in mice produceneurological outcomes that mimic the disease in all essentialfeatures. Thus, the murine model of ALS-PDC meets thecriterion of etiological validity.

Construct validity is dened as the accuracy with which atest measures what it is intended to measure. Any animalmodel of a progressive neurological disease should provide

predictable time-dependent losses of neural cells in thesame CNS regions affected in the human disease. The ALS-PDC model satises this criterion since it demonstratesprogressive neuronal loss in regions of CNS accompaniedby appropriate behavioral dysfunction, both resemblingmeasurable features of ALS-PDC. In addition, the phenom-enology is robust in that it has been routinely observed inmultiple batches of animals and displays the range of motorand cognitive outcomes that comprise the diverseexpression of the various sub-disorders in ALS-PDC, i.e.ALS, PDC and the combined symptoms.

The nal standardpredictive validityis dened as theability of a test to predict a criterion that is of interest tothe investigator [46]. In any neurodegenerative disease, themajor criterion of interest is a progressive degeneration of

specic neuronal subsets. For example, in Parkinsonsdisease, the major neuronal degeneration is in the substantianigra and striatum; in ALS, degeneration of upper and/orlower motor neurons of the brain/spinal cord; AD istypied by degeneration of cortical neurons and corticalthinning along with the appearance of NFT and/or amyloidplaques. Our model of ALS-PDC meets this criterion aswell: Mice fed with washed cycad our display progressivecognitive and motor behavioral decits as well ascorresponding CNS pathologies. In addition, a clearly pre-dictive feature of our model is the olfactory sensorydecit and disruption of the morphology of olfactoryglomeruli. These data predict similar decits in humanAD, PD, ALS, and ALS-PDC, features which are nowreported in Refs. [5153] .

8. Geneticepigenetic interactions in the cycad model

Our results have clearly demonstrated that an exogenousneurotoxin contained in cycad can induce features of ALS-PDC in a mouse model. These results lend strong support tothe notion that many of the features of the human disease, aswell as similar age-related neurodegenerative diseases,could have as their basis an environmental toxin to which

various fractions of the population are exposed andsusceptible. The notion that environmental toxins could beprimary etiological factors has long been considered forvarious neurological diseases and a recent study on identicaltwins lends increasing support to the notion that environ-ment, rather than a simple genetic factor, is crucial (see Ref.[54]). Nevertheless, familial forms of these diseases existand in such cases there is abundant evidence for geneticcausality of a limited scope. Animal models of geneticfactors involved in human neurological diseases haveprovided much information about potential mechanismsleading to cell death. For example, transgenic miceover-expressing b -amyloid show cognitive losses andneuronal damage similar to AD and mutant superoxidedismutase (mSOD1) mice expressing a toxic gain of


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function mutation for SOD show a progressive degenerationof motor neurons.

Given the above data, it is too simplistic to view any of the sporadic forms of neurological disease as solely theresult of either genes or environment. In fact, a growingnumber of investigators have concluded that the intersectionof genetic-epigenetic factors is involved. Fig. 3 illustratesthis concept, showing the intersection of genetic suscepti-bility factors with environmental toxins, along with age asthe critical third variable. This last factor is crucial,especially given that the diseases under discussion in thisarticle all occur within specic age ranges, usuallybeginning in late middle age.

We have begun to examine gene-environment inter-actions using our cycad mouse model due to the ability of

this model system to reliably induce neurodegeneration inspecic neural subsets. We have now combined cycadfeeding with two genetic variants. First, APO E transgenicmice have been used to explore the interaction of cycadtoxins with genetic susceptibility co-factors. APO Eproteins are involved in cholesterol handling and thevarious allele variants have been implicated in AD, ALS,and ALS-PDC [55]. Second, transgenic mice over-expres-sing mutant human SOD (mSOD1) have been used toexamine 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 displaysignicant behavioral decits, in marked contrast to cycad-fed wild-type (WT) APOE mice which showed signicant

decits across a range of functions [57]. Histology showedcell death in the substantia nigra, hippocampus, andstriatum in cycad-fed WT mice, but not in cycad-fedAPOE KO mice. Cycad-fed APOE KO mice also showedincreased cholesterol levels without displaying cycad-induced damage to heart or liver [57]. (Experiments inwhich cycad will be fed to mice expressing specic APOEisoforms (E2, E3, and E4) are currently underway.) Thesedata clearly demonstrate that certain genetic susceptibilityfactors can increase or decrease sensitivity to toxinexposure, reinforcing the notion that the interplay of genes and environment is likely part of the overallconstellation of factors leading to neurodegenerativedisease. With regard to this last point, additional complex-ity is certain to arise in future experiments as we examine

the complete temporal sequence of events from initial toxicinsult 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 simplythe sum of cycad effects combined with those of themutation [58]. For example, measurements of leg exten-sion, an index of spinal motor neuron integrity, showedthat mSOD mice fed cycad had a move rapid decline thaneither mSOD mice or cycad-fed wild type mice. Measure-ments of latency to fall on a rotarod test gave a verydifferent picture with cycad-fed mSOD mice performingbetter than mSOD mice not exposed to cycad. These dataare preliminary and await conrmation, but highlight again

the notion that gene-environment interactions are likely tobe complex.

9. Time line of neurodegenerative eventsin the cycad model

As described above, our studies in cycad-fed mice haveutilized a battery of behavioral, biochemical, and morpho-logical measures to create a working time line for theemergence of behavioral and pathological outcomes begin-ning with the initial toxin insult and progressing through toanimal death. The data clearly reveal that the variousbehaviorsare impacted at different times following exposureto cycad toxins. We do not yet have correspondingbiochemical and histological studies for each time point,but these data arenowbeginning to emergefrom ourongoingstudies. Forexample, we nowknow that the down-regulationof glutamate transporter subtypes is a relatively early event,likely occurring long before cell death [44]. Placed incontext, the alterations in protein kinase C we have noted inprevious experiments, are likely to occur even earlier andmaypossiblybe involved in abnormal phosphorylationof thetransporter leading to down-regulation and loss of function[44] . Current studies will extend this timeline frominitial insult, through the period of overt expression of neurological outcomes, and the stages leading to neural celldeath. Fig. 4 illustrates our progress to date and provides

Fig. 3. Potential synergies of causal and risk factors in sporadicneurological disease. Causal factors involved in such diseases may reectexposure to toxin(s) that can arise from the environment or as a result of individual biochemical processes. In the former case, the toxins may besyntheticor naturallyoccurring. The toxic factor is represented by the set onthe left side of the diagram. The range of toxins effects run from left toright as low to high. Intersecting this is a set consisting of a geneticsusceptibility factors that could arise due to genetic polymorphism inefciency of detoxication mechanisms (from right to left, expressed ashigh to low). Genes coding for transport proteins (e.g. APO E alleles)could also be involved. The intersection of these two sets describes theindividuals who may be at risk of developing the neurological disorder.

Note that the intersecting region can increase or decrease depending onstrength of either variable. Intersecting these two sets is the variable of agewith the risk factor increasing from young to old (bottom to top).


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a current interpretation of where the various behavioral andpathological events may be found along the timeline.

Once the model timeline is completed, we will be in aposition to attempt therapeutic interventions in our treatedanimals at each stage of the disease process to preventfurther deterioration. Even if successful, however, ulti-mately we will need to transpose these data to the humanpopulation in order that such treatments can be directed tothe right target at the precise time to have the greatestimpact. The template matching between data from animalmodels and human disease states, especially preclinicalstates, remains the greatest single challenge for treatingneurological disease patients.

10. Template matching to human neurological disease

What is actually known about human neurological diseaseprogression? A search of the literature reveals that the ratesof decline for the various neurological diseases vary

quantitatively for each disease post clinical diagnosis, butthat the decline function is usually linear. For example inALS, progression is linear for the decline of motor neurons[59] asis the riskof death [60] , but these measures depend onthe timeof diagnosis [61] . For PD, there isa variablecourseof progression of different PD symptoms depending on age of onset, but these are always linear [6264] . For AD, diseaseprogression measured by cognitive decline varies dependingon sexandageof behavioral onset [65] . Unfortunately, llingin more details is unlikely to offer much in the way of treatment options. Of greater concern, there seems to be verylittle data about pre-clinical stages of these diseases, whichmay show different rates of decline compared to post-diagnosis. For example, loss of function (strength andfunctional activities) is linear, but the loss of motor neurons

may be exponential with an initial rapid fall that precedesdiagnosis followed by a more linear motor neuron loss as thedisease progresses [66] . However, it is during the pre-clinicalstage that thehopefor effectiveprophylaxisor early treatmentexists.

This pre-clinical gap is where animal models canmake the greatest contribution, for by extending thetimeline backwards to disease-initiating factors theyallow us to focus attention on the earliest potentiallytreatable stages of each disease. Although results arescattered and incomplete, some attempts have been madein this regard. For example, in the amyloid b model of AD,researchers have shown an exponential increase in amyloidb deposits [67]. Similarly, mSOD mice used to model ALSshow exponentially increasing microglial activation from 0to 120 days [68]. Some examples from the literature aresummarized in Table 2 . In regard to our own model, cycad-fed mice show exponential decays in motor and cognitivefunctions, interspersed with what appear to be temporarysurges in behavioral compensation (see Refs. [7,69] ).

In Fig. 5 we merge the two data sets, including the ALS-PDC model system data with those from studies of humanage-dependent neurological disease. Note that the former

Fig. 4. ALS-PDC mouse model timeline of various behavioral andpathological events. Events described are from data collected from cycad-fed mice at the time point in which the event falls. Progressing Behaviourlists events occurring during the time from start of feeding to sacrice. TheSacrice column describes events that were found post mortem in CNStissue. Predicted Progression describes hypothetical progression of thecycad-induced outcomes. Arrows indicate predicted continuations ( ! ) or

predicted originations ( ) of the events described. " : increasinglevels/amounts. # : decreasing levels/amounts. Abbreviations: MNs:ventral horn spinal cord motor neurons; NFT: neurobrillary tangles.

Table 2Disease progression of human and animal modeled neurodegenerative disorders

AD PD ALS

Human (post-diagnosisprogression)

Disease progression roughlyconstant with time [65]

Variable course of progressionof different PD symptoms dependingon age of onset, but always linear [62]

Linear decline of motor neurons [59]

Constant risk of death ( Fig. 3a ) [63] Constant risk of death, but dependingon time of diagnosis [60]

Dopaminergic degeneration in PDappears to slow down during courseof the disease [64]

Mouse models (entiredisease progression)

Exponential increase of Amyloid B deposits inPS1 APP transgenic ADmouse [67]

Exponentially decreasing risk inchemically induced model of PD(Fig. 3D) [63]

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

Disease progression ratesare comparedfor Alzheimers disease (AD), Parkinsons disease (PD)and amyotrophiclateral sclerosis (ALS).Human disease ratesare determined from beginning of behavioral symptoms, while animal models measure progression from onset to death.


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starts from the initial insult and moves forward in time to theend state condition, while the latter attempts to reverseengineer the earliest stages from the end state. Neither dataset alone is sufcient, but the hope is that the future successof our model will allow us to ll in the enormous gaps thatnow exist concerning the timeline of pathological events inhuman neurological diseases.

As discussed above, the problem is that we have notimeline to speak of for tracing the course of age-dependenthuman neurological diseases. How, for example, can weknow who is pre-symptomatic for any of these diseases,especially any of the sporadic varieties? In principle, variousbrain scans or biomarkers could be widely employed, but therst is not economically feasible for the population at large;the second would depend on the identication of crucialmolecules, most of these still unknown. The necessarycrossover and template matching from an animal model tohuman neurological disease states is a twofold process. First,we have to be able to place what we know from our modelsystem into a clear description of events in all fourdimensions. Second, armed with this information, we canthen attempt template matching to pre- and post-clinicalhuman patients. This procedure may be a time consumingone, yet offers, in our view, the only realistic possibility forprevention and early treatment of human neurologicaldisease. We discuss a potential strategy in more detail below.

11. The mouse model of ALS-PDC: implications forprophylaxis and for halting disease progression

Our work has demonstrated the following: First, we havebeen able to show that a toxin contained in cycad is able tokill neurons. Mice fed with washed cycad develop the samebehavioral 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 longprocess of dening the various stages leading from toxicinsult to neurodegeneration.

Each of these outcomes has potentially enormousimplications for detecting and treating neurological disease.The identication of potential toxins should now spark asearch for these molecules and molecules sharing theirmode of action in the human environment. The isolation andpartial characterization of the toxicity of various sterolglucosides as the toxins giving rise to ALS-PDC maysuggest that an important future goal is the identication of the sources of these toxins. Tracing such toxins wouldnaturally include a search for them in food products otherthan in cycad. For example, soybeans may contain sterolglucoside levels greater than those of cycad (Soybean our:214.9 mg/g [70]; Cycad: 11.584.5 mg/g [28,43] ), althoughwhether processed soy does so as well is still an unansweredquestion. In addition, sterol glucosides may be present in

other sources, i.e. a data base search reveals that sterolglucosides are found in tobacco and survive in tobaccosmoke [71,72] . A recent report also shows that Helicobacter pylori bacteria make a similar sterol glucoside, specically acholesterol glucoside, [73,74] (note that Khabazian et al.[43], found cholesterol glucoside to be extremely neurotoxicin vitro). H. pylori infections are associated with increasedrisk for PD [75] and this potential link to PD, and thepossible relation to other neurological disorders, should beexamined in greater detail.

Sterol glucosides are only one type of molecule able toinduce the types of neurodegeneration seen in our modeland in human neurological disease. Further, the sterol

glucosides identied by us may contribute only a smallfraction of total disease cases. From this, two possibilitiesarise: rst, structurally dissimilar molecules may havesimilar mechanisms of action. Second, sterol glucosidesmay interact synergistically with molecules having verydifferent mechanisms of action on neurons. In the lattercase, we consider the possibility of such interactionsbetween excitotoxins and oxidative stress (see Ref. [76]).Overall, the identication of potential neurotoxins mayserve as a rst means of prophylaxis since if we can detectsuch molecules in the environment, we may be able to avoidthem.

Of equal importance, the notion that certain geneticsusceptibilities may be crucial, ties in with the identicationof 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 putativetoxin could interact. For example, our identication of sterolglucosides, including cholesterol glucoside, as potentialneurotoxins in neurological disease is directly relevant tothe notion that APO E allele variants can contribute to theexpression of such diseases. Similarly, genes coding for thesynthesis or modication of other sterols, e.g. the variousCYP genes [77,78] , for example, may be important. Inaddition, genes that control enzymes involved in thesynthesis or degradation of sterol glucosides could becrucial [79,80] . The identication of human genetic proles,

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


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especially as they relate to environmental toxins, may serveto identify those persons at future risk of developing orexpressing the disease. Prophylaxis may be achieved byidentication of the putative toxin or by the identication of those whose genetic susceptibility makes them vulnerable tothat toxin.

As valuable as prophylaxis would be to those not yetaffected, what does any of this imply for those at early andmiddle stages of exposure and presumed emerging neuraldamage? How are these individuals to be identied in orderthat targeted therapeutics halt the biochemical cascadesfrom culminating in neurodegeneration and the clinicalmanifestation of disease? The answer to this must arise froma thorough understanding of the stages of the diseaseprocess and, as discussed, this can only occur via a

comprehensive animal model that combines behavioral,biochemical, and histological analyses over an extendedtime period. In addition, an understanding of the full four-dimensional aspects of the disease process will allowtargeted therapeutics to be applied to halt degenerativecascades. For example, early glutamate transporter abnorm-alities [44] created by excessive kinase activity might bereversed by either a selective block of that kinase or aninduced up-regulation of the transporter. The timelyapplication of therapeutic agents, at the right place andtime, will be key components of a successful treatmentparadigm.

The challenge, however, is to extrapolate the animal four-

dimensional data to the pre-clinical human population. Weenvision a process something like the following: The mousetimeline provides not only sequenced events leading toneurodegeneration, but also comparative behavioral dataand, perhaps, the identication of various biomarkers forearly stages of the disease process. Working backwards frompatients currently diagnosed with the various diseases, wecan attempt strong correlations to earlier pre-clinicalbehavioral changes. These, in turn, may lead to attempts toidentify such behavioral outcomes in pre-clinical popu-lations. A good example of this process would be the use of the apparent early olfactory disturbance in PD [51,52] toplace these individuals into the correct position in thetimeline. Behavioral indices of altered neural function mightthen trigger the search for identied biomarkers, the lattersymptomatic of unique changes within the nervous system atparticular time points. MRI or other imaging methods couldthen be employed to conrm changes to neural morphology.As above, template matching between human patients andanimal models may allow a blockade of the neurodegenera-tive cascades at an early stage of the disease progression,when most behavioural function is still intact.

12. Conclusion

ALS-PDC is a unique disease that displays aspects of themajor progressive neurodegenerative diseases, AD, PD and

ALS. The exceptional features of ALS-PDC may suggestthat AD, PD, and ALS share some common features andmay arise in part due to common etiologies. The ALS-PDCbehavioral and neuropathological outcomes can be repro-duced in an animal model based on the consumption of toxins contained in cycad seeds. The data obtained by thismodel include the possible identication of the putativeneurotoxin as well as a preliminary understanding of thetemporal progression of neurodegeneration followingexposure. In addition, we have begun to explore aspectsof gene environment interactions as determinants of neurodegeneration. The insights gained by use of thismodel may allow for future prophylaxis and treatment of human neurological diseases.

Acknowledgements

This work was supported by grants from the ALSAssociation, Scottish Rite Charitable Foundation of Canada,Natural Science and Engineering Research Council of Canada, and the US Army Medical Research and MaterielCommand (#DAMD17-02-1-0678) (to CAS). The authorsthank 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 glutamatetransporter labeling, respectively. The authors also thank H. Bavinton, C Melder, and M. Wong for comments on themanuscript. Cycad seeds were provided by our colleagueson Guam, Drs U. Craig and T. Marler to whom we are verygrateful. This review acknowledges the unique contri-butions of the late Dr L.T. Kurland.

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