Experimental Neurology 176, 1–11 (2002)doi:10.1006/exnr.2002.7940

REVIEW

Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex:Transgenic Mice Provide Insights into Mechanisms Underlying

a Common Tauopathy in an Ethnic Minority on GuamJohn Q. Trojanowski,1 Takeshi Ishihara, Makoto Higuchi, Yasumasa Yoshiyama, Ming Hong,

Bin Zhang, Mark S. Forman, Victoria Zhukareva, and Virginia M.-Y. LeeThe Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine,

The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received February 4, 2002; accepted April 11, 2002

Intracytoplasmic filamentous tau inclusions areneuropathological hallmarks of amyotrophic lateralsclerosis/parkinsonism–dementia complex (ALS/PDC) of Guam and the defining lesions of other neu-rodegenerative disorders known as tauopathies.Here we review current insights into the cell andmolecular neuropathology of ALS/PDC, a commontauopathy in the Chamorro population on Guam. Wealso summarize recent advances in understandingthis disorder through studies of transgenic (Tg)mouse models of this tauopathy. Briefly, overexpres-sion of human tau isoforms in the central nervoussystem of Tg mice resulted in a neurodegenerativetauopathy with a phenotype similar to ALS/PDC.Specifically, argyrophilic, congophilic, and tau im-munoreactive inclusions accumulated with age incortical and brainstem neurons of these mice, butthey were most abundant in spinal cord neurons,and the inclusions contained 10- to 20-nm tau-posi-tive straight filaments. There also was extensive gli-osis in spinal cord associated with axonal degener-ation in the ventral roots, while remaining axons inspinal nerves showed a loss of microtubules and re-duced fast axonal transport. With advancing age,these Tg mice showed increasing motor weakness,and this was accompanied by a progressive increasein the phosphorylation and insolubility of brain andspinal cord tau proteins. Thus, tau Tg mice recapit-ulate key phenotypic features of ALS/PDC neuropa-thology in an ethnic minority on Guam, and theseanimal models provide new opportunities to

1 To whom correspondence should be addressed at Center for Neu-rodegenerative Disease Research, Maloney 3, HUP, 3600 SpruceStreet, Philadelphia, PA 19104-4283. Fax: (215) 349-5909.

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discover novel therapies for this and relatedtauopathies. © 2002 Elsevier Science (USA)

INTRODUCTION

Tau is an abundant microtubule-associated protein inthe central nervous system (CNS) that is implicated inthe pathogenesis of amyotrophic lateral sclerosis (ALS)/parkinsonism–dementia complex (PDC) of Guam (ALS/PDC), Alzheimer’s disease (AD), frontotemporal demen-tia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), corticobasaldegeneration (CBD), and a number of other neurodegen-erative diseases known as tauopathies (see Table 1 andRefs. 6, 14, 23, 28, 46 for recent reviews). Like othertauopathies, Guam ALS/PDC is characterized neuro-pathologically by numerous inclusions similar to AD neu-rofibrillary tangles (NFTs) formed by aggregated pairedhelical filaments (PHFs) and/or straight filaments. Theseinclusions are composed of aberrantly phosphorylatedtau proteins (PHF-tau) in selectively vulnerable neuronsand glial cells throughout widespread regions of the CNS(for reviews, see Refs. 12, 14, 28, 44).

Six alternatively spliced tau isoforms are expressedprimarily by neurons in the adult human CNS and arelocalized predominantly in axons, although glial cellsalso contain small amounts of tau (reviewed in 6, 23,46). Tau proteins bind to microtubules (MTs), promotethe assembly of MTs, and stabilize MTs in the poly-merized state, but the formation of PHF-tau results ina loss of these important functions. Moreover, unlikenormal tau, PHF-tau is insoluble, accumulates in thesomatodendritic domain of neurons, and assemblesinto filaments that aggregate as NFTs in Guam ALS/PDC, AD, and related tauopathies (8, 14, 23, 28, 46).However, in addition to PHF-tau, other cytoskeletalproteins, i.e., neurofilament (NF) subunits, also are

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found in many NFTs as in ubiquitin, although thepathological significance of these tangle-associatedcomponents is unclear (6, 23, 46).

The massive degeneration of neurons and extensivegliosis associated with the progressive accumulation ofPHF-tau lesions provided circumstantial evidence im-plicating filamentous tau pathology in the onset/pro-gression of neurodegenerative disease (6, 23, 46). How-ever, the discovery of multiple pathogenic mutations inthe tau gene of many distinct FTDP-17 families dem-onstrated directly and unequivocally that tau abnor-malities cause neurodegenerative disease. Thesepathogenic FTDP-17 mutations are located at topo-graphically distinct sites in exons and introns of thetau gene including exonic missense substitutions, anin-frame deletion, and intronic substitutions (6, 23,46). Significantly, emerging evidence suggests that to-pographically separate mutations cause FTDP-17 bydifferential mechanisms that specifically alter thefunctions or levels of tau isoforms in the CNS (17).

ALS AND PDC IN THE ETHNIC CHAMORROMINORITY POPULATION OF GAUM

Guam is the largest island in the Mariana grouplocated in the Western Pacific, and it had been welldocumented in the late 1940s that there was a veryhigh incidence of neurodegenerative diseases on Guamand others of the Mariana Islands (reviewed in 8, 12–14, 27, 28, 44). These disorders were characterized by

two main clinical phenotypes, and they affected mainlyor exclusively the indigenous Chamorro population ofGuam. The two major Chamorro disorders were de-scribed by investigators in the 1940s–1950s, and theybecame known as ALS and PDC in the scientific liter-ature, but they were referred to as lytico and bodig,respectively, by the indigenous Chamorros on Guam.While Guam ALS is clinically indistinguishable fromALS in the continental United States and elsewhere inthe world, Guam PDC is a distinct neurodegenerativedisorder wherein parkinsonism and dementia cooccurin affected Chamorros. As a result of the initial effortsby investigators to define the prevalence of ALS andPDC in the 1950s and early 1960s on Guam, it wasshown that that these disorders were exceptionallycommon with a prevalence of �120–140:100,000among the indigenous Chamorro population. However,subsequently, from 1960 to 1983, the prevalence ofALS appeared to decrease as did that of PDC, albeit toa lesser extent. Moreover, this was associated with adelay in the age at onset of both disorders relative topatients affected by these diseases in the 1950s, butthe reasons for these changes in the incidence, preva-lence, and onset of these diseases remain unexplained.Further, despite clinical similarities between theseChamorro disorders and their counterparts outsideGuam (1, 8, 24, 27, 30, 44), the neuropathology ofGuam ALS and PDC is distinct. Guam ALS and PDCare characterized by widespread NFTs containing in-soluble aggregates of tau, which are morphologicallyand biochemically identical to those found in AD, withonly a small percentage (�25%) of affected patientsshowing evidence of amyloid deposits similar to thosefound in AD (3, 4, 12–16, 26, 28, 36, 39, 40) (see Fig. 1for representative examples of the tau pathology inGuam ALS and PDC). Nonetheless, there are accumu-lations of alpha-synuclein filaments in Lewy bodies,especially in neurons of the amygdala in PDC similarto sporadic and familial AD and Down’s syndrome pa-tients with AD (7, 45).

However, there are differences between the tau pa-thology in these Guam disorders and that in AD orother tauopathies including the phosphorylation stateof tau, the morphology of PHF-tau filaments, and thetopography of tangles in different regions of the CNS(15, 16, 25, 26, 28, 31, 35, 38, 40). Further, the distri-bution of NFTs and neuron loss in the two major formsof Chamorro neurodegenerative disease differs, but theburden of the neuropathology in Guam ALS and PDCroughly reflects the clinical manifestations of each dis-ease (8, 12–24). For example, in ALS, the major neu-ropathology is in the caudal neuraxis, especially spinalcord, wherein there is extensive neurodegeneration ofspinal motor neurons in the anterior horn accompaniedby accumulations of NFTs in surviving spinal cordneurons with lesser involvement of other more rostralbrain regions. In contrast, NFTs accumulate in rostral

TABLE 1

Major Neurodegenerative Tauopathies

Sporadic/Familial Alzheimer’s diseaseAmyotrophic lateral sclerosis/parkinsonism–dementia

complexArgyrophilic grain dementiaCorticobasal degenerationDementia pugilisticaDiffuse neurofibrillary tangles with calcificationDown syndromeFrontotemporal dementia with parkinsonism linked to

chromosome 17Gerstmann–Straussler–Scheinker diseaseHallervorden–Spatz diseaseInclusion body myositisJakob–Creutzfeldt diseaseMultiple system atrophyNiemann–Pick disease type CPick’s diseasePrion protein cerebral amyloid angiopathyProgressive supranuclear palsySubacute sclerosing panencephalitisTangle-predominant Alzheimer’s disease

Note. Sporadic and hereditary neurodegenerative diseases charac-terized by filamentous tau pathology in brain and spinal cord. Pro-totypical tauopathies wherein glial and/or neuronal tau inclusionsare the sole or predominant CNS lesions are indicated in boldfacetype. See text for further details.

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portions of the CNS in PDC, especially in hippocam-pus, in other limbic regions, and in neocortex. Finally,while Guam ALS and PDC syndromes can be distin-guished in affected Chamorros, not uncommonly bothdisorders may cooccur in the same patient and thecombined condition is known as Guam ALS/PDC (re-viewed in 14).

While earlier evidence suggested a declining inci-dence of ALS and PDC on Guam through the 1970s and1980s, more recent studies demonstrate the persistentoccurrence of PDC among Chamorros on Guam, albeitabout a decade later in their lifespan, while cases of

ALS continue to diminish in frequency (8, 27, 44).These changes in the expression of Chamorro neurode-generative diseases are likely to reflect an interactionof environmental and genetic factors among Chamor-ros living on Guam. However, despite the efforts ofmany investigators to identify environmental factorsthat might cause ALS and PDC (e.g., toxins found inflour prepared from cycad nuts that are part of the dietof indigenous Chamorros or the high levels of alumi-num found in the water on some parts of Guam), it hasbeen difficult to establish an environmental cause ofthese Chamorro disorders (5, 32). Moreover, while ev-

FIG. 1. Tau pathology in Guam ALS and PDC. (A) NFTs in neurons of the hippocampal formation of Guam PDC patient; immunohis-tochemistry with phosphorylation-dependent anti-tau antibody PHF1. (B) NFTs in neurons of amygdala of Guam PDC patient; immuno-histochemistry with phosphorylation-dependent anti-tau antibody AT8. (C, D) Tau-positive inclusions in astrocytes of amygdala of GuamPDC patients, many with the morphology of hazy, granular astrocytes similar to those described by Oyanagi et al. (31); immunohistochem-istry with AT8. (E, F) Tau inclusions in neurons of anterior horn in Guam ALS patients: E, PHF1; F, AT8. Scale bar, 80 �m (A), 40 �m (C),20 �m (B, D, E, F). (G) Insoluble fractions (PHF-tau) from AD and Guam ALS/PDC patients show similar patterns of tau isoform composition.Sarkosyl-insoluble fractions (PHF-tau) from gray and white matter of different brain regions from a Caucasian patient with AD (left) and aGuamanian ALS/PDC patient (right) were dephosphorylated with Escherichia coli alkaline phosphatase, resolved by SDS–PAGE, andimmunoblotted with anti-tau antibodies T14 and T46. Recombinant tau isoforms are as indicated. Fr, frontal lobe; Te, temporal lobe; Pa,parietal lobe; Oc, occipital lobe; Cc, cerebellum; G, gray matter; W, white matter; rTau, recombinant tau.

3MODELS OF NEURODEGENERATIVE TAUOPATHIES OF CHAMORROS

idence to suggest that genetic factors which predisposeto AD might also predispose to Guam ALS and PDC isinconclusive, the discovery of pathogenic tau gene mu-tations in FTDP-17 stimulated efforts to identify sim-ilar mutations in Chamorros (8, 11, 33).

Indeed, the Chamorros appear to be a tractable pop-ulation for elucidating genetic causes of disease be-cause they are an inbred minority group that haveresided on Guam for centuries, although the popula-tion has fluctuated over time (8, 33). For example, inthe 17th Century, the population decreased from about70,000 to fewer than 2000 due to natural disasters(e.g., typhoons and earthquakes that still occur in theMariana Islands), conflicts with Spanish colonialists,and the arrival of new diseases such as smallpox.These aspects of the history of the Chamorros in theMariana Islands raise the possibility that foundergenes may account for the uncommonly high preva-lence of neurodegenerative disease among them. None-theless, this is confounded by the admixture of othergenes due to intermarriage of the Chamorro populationwith Filipino and Spanish settlers and to a lesser de-gree European whalers and traders during the 17th–19th centuries. While no pathogenic tau gene muta-tions to account for Guam ALS and PDC have beenreported to date, it is plausible, given the large familyclusters of ALS and PDC among Chamorros, that thereare genetic risk factors linked to the tau locus or a taurisk factor allele (e.g., the 142 or A0 allele) involved inthe pathogenesis of these diseases (33). However, fur-ther complicating elucidation of the underlying basisfor ALS and PDC is the emergence more recently of anAD-like dementia among elderly Chamorros that isreferred to as Mariana dementia (8). Although cases ofdementia had been reported earlier on Guam, morerecent studies suggest a high prevalence of PDC and ofdementia in Chamorros over the age of 40 years, butmore work is needed to define the nature of the demen-tia in elderly Chamorros. Indeed, there is a remarkablylow frequency of the Apolipoprotein E �4 allele inChamorros (around 4–7%) despite the fact that thisallele is a defined risk factor for AD in many popula-tions outside Guam (33). Thus, there are intriguingclues suggesting a powerful but unexplained mecha-nistic interaction between the genes of the Chamorropopulation of Guam and the environment in the Mari-ana Islands to account for the shifting incidence andphenotypic manifestations of neurodegenerative dis-eases affecting this unique minority population. Fur-thermore, in light of the similarities between GuamALS/PDC and other tauopathies, it is likely that elu-cidation of this perplexing enigma will provide impor-tant clues into mechanisms underlying the more com-mon neurodegenerative disorders elsewhere in theworld.

ALS/PDC-LIKE PHENOTYPE IN TAUTRANSGENIC MICE

Building on earlier efforts to model tauopathies (2, 9,10, 18, 23), we initiated studies to test the hypothesisthat neurodegenerative disease can result from alteredexpression levels of normal tau isoforms by generatingtransgenic (Tg) mice that overexpress the shortest hu-man brain tau isoform (“fetal tau”) in CNS neurons(19–21). In our initial studies, we reported that theseTg mice develop progressive age-dependent accumula-tions of intraneuronal filamentous inclusions accompa-nied by neurodegeneration, gliosis, and tau proteinabnormalities similar to Guam ALS/PDC (20). The suc-cessful generation of Tg mice that model a neurodegen-erative tauopathy with the phenotype of ALS/PDCopened the way for producing additional Tg mice thatmodel other human tauopathies. Strategies for the de-sign and evaluation of these Tg mouse models wereextended in subsequent studies by our group (19, 21)and by other investigators (9, 18, 23). Indeed, the grow-ing literature on tau Tg mice and other animal modelsof tau pathologies were the subject of two recentvery comprehensive reviews (9, 18). Thus, we focusthis review on the ALS/PDC-like neurodegenerativetauopathy in lines of tau Tg mice that we generatedand characterized and on the effects of crossing thesemice with NF protein knock-out mice. We also considerhow these and other tau Tg mice can be exploited inresearch to develop a better understanding of thepathogenesis of these disorders and more effectivetherapies for Guam ALS/PDC and related tauopathies.

Generation of Tg Mice That Overexpress the ShortestHuman Tau Isoform

To generate Tg mice expressing human tau, a cDNAcorresponding to the shortest human brain tau isoform(T44, also known as “fetal tau”) was cloned into anexpression plasmid (MoPrP.Xho) containing the pro-moter, 5� intronic, and 3� untranslated sequences of themurine prion protein gene (20). We identified threestable Tg lines that were shown by Western blot anal-ysis to variably overexpress human tau and transmitthe gene in a Mendelian manner. Quantitative West-ern blot analysis of total protein from Tg mice andwild-type (WT) littermate controls before and after de-phosphorylation showed that tau proteins from allthree Tg mouse lines, but not from the WT mice, weredetected by T14, a human tau-specific monoclonal an-tibody (MAb). Western blots with this same MAb alsoshowed that the dephosphorylated tau protein from theTg mice comigrated with recombinant fetal human tau.Using a polyclonal antibody (17026) that recognizeshuman and mouse tau in quantitative Western blotstudies, we showed that the heterozygous Tg mouselines 7, 43, and 27 overexpressed tau proteins at ap-

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proximately 5-, 10-, and 15-fold higher levels, respec-tively, than endogenous mouse tau. However, expres-sion varied throughout the CNS of the Tg mice. Forexample, neocortex, hippocampus, brainstem, and cer-ebellum expressed comparable levels of Tg tau, butspinal cord expressed only �60% of the tau levels seenin these regions. Since Tg line 27 mice were not viablebeyond 3 months, and homozygous Tg mice generatedfrom each of these three lines of Tg mice died in uteroor within 3 months postnatal, the observations sum-marized below come from studies conducted on 1- to12-month-old heterozygous tau Tg mice from lines 7and 43.

Tau Tg Mice Acquire CNS Tau Inclusions with AgeSimilar to Those in ALS/PDC

Histological analysis of Tg mice and their WT litter-mates from lines 7 and 43 between 1 and 12 months ofage revealed a widespread expression of human tau inneurons and their processes throughout the CNS of theTg, but not the WT, mice, which remained constant atall ages examined (20). In Tg mouse spinal cords, T14-positive spheroid-like intraneuronal inclusions wereobserved at 1 month, and the size and number of theseinclusions increased up to 6–9 months. Notably, manyvacuoles the same size or larger than the inclusionswere also observed in the older Tg mice, which mayreflect degeneration of affected axons. The inclusionswere about the size of spinal cord neurons, and somearose within proximal axons of spinal cord neurons.Although they occurred in gray and white matter at allspinal cord levels, the inclusions were most frequent atthe gray–white junctions (see Fig. 2 for representativeexamples of the tau pathology in these Tg mice).

Spinal cord sections were probed with a panel ofantibodies to tau and other neuronal cytoskeletal pro-teins, and the inclusions were immunostained by aconformation-dependent antibody commonly used todetect tau protein found in PHFs (i.e., Alz50) and byadditional phosphoryation-dependent tau antibodiesincluding PHF1 (phosphoserines 396 and 404, number-ing according to the largest human brain tau), PHF6(phosphotheronine 231), T3P (phosphoserine 396), AT8(phosphoserines 202 and 205), AT270 (phosphothero-nine 181), and 12E8 (phosphoserine 262). Therefore,these lesions contain hyperphosphorylated tau similarto PHF-tau in AD and Guam ALS/PDC. Significantly,these inclusions were also stained strongly with anti-bodies to the low (NFL)-, middle (NFM)-, and high(NFH)-molecular-weight NF proteins. Both phosphor-ylated and nonphosphorylated NFM and NFH wereobserved in these lesions. Indirect immunofluorescencedouble-labeling with antibodies 17026 and RMdO9confirmed the colocalization of tau and NFs in theseinclusions. In addition, anti-tubulin antibodies immu-nostained these inclusions.

In the brains of the Tg mice, tau-positive intraneu-ronal aggregates were also detected, but they weresmaller and developed later than the spinal cord inclu-sions. They were first seen in the pontine neurons of1-month-old animals and emerged in the cerebral cor-tex at about 6 months of age. The immunohistochemi-cal profile of these brain aggregates was similar to thatof the spinal cord lesions; i.e., they contained hyper-phosphorylated tau, all three NF subunits, and tubulinepitopes. However, the morphological features of theseinclusions indicate that some are variants of the spinalcord axonal lesions, while others occur in the somato-dentritic compartment of cortical neurons and resem-ble NFTs and dystrophic neurites. Notably, the brainand the spinal cord inclusions were positively stainedby the Bodian silver method, similar to human NFTs,but were Thioflavin S-negative, and they were notstained by antibodies to alpha-internexin, peripherin,ubiquitin, and synucleins. Line 43 expressed higherlevels of human tau than line 7 and similar tau-richinclusions were observed in the spinal cord and thebrain in an age-dependent manner, but they werelarger and more abundant than those in line 7. Thisindicates that the accumulation of these tau-rich le-sions in the Tg mice is transgene dose and age depen-dent.

Transmission electron microscopy (EM) studies ofthese inclusions revealed tightly packed aggregates ofrandomly arranged 10- to 20-nm straight filaments inmyelinated spinal cord axons of Tg, but not WT, mice(20). These aggregates were found in �30% of myelin-ated and unmyelinated axons, ranging from 200 nm to20 �m in diameter and some inclusions nearly filledthe axon. Mitochondria were trapped within occasionalaggregates. Immuno–EM studies showed that the fila-ments were immunolabeled by antibodies to tau, NFs,and tubulin.

Since the argyrophilic filamentous lesions of thesetau Tg mice were concentrated in the spinal cord andbrain stem, similar to those observed in some ALS/PDC, PSP, CBD, and FTDP-17 patients, we directlycompared the tau inclusions detected in the mice withthose in the human diseases. (20). Significantly, in theALS/PDC spinal cord, NF immunoreactivity colocal-ized with tau in many of the inclusions. These data,taken together with the findings described above, sug-gest that these tau Tg mice develop a neurodegenera-tive disease that recapitulates the hallmark lesions ofGuam ALS/PDC and other related human tauopathies.

Insoluble Tau Protein Progressively Accumulates inthe CNS of Tau Tg Mice

To determine whether tau becomes progressively in-soluble in the tau Tg mice in an age-and disease-de-pendent manner similar to that of the human tauopa-thies, we analyzed the solubility of tau from both brain

5MODELS OF NEURODEGENERATIVE TAUOPATHIES OF CHAMORROS

FIG. 2. Pathological and biochemical characteristics of Tg mouse model of ALD/PDC. (A) Spinal cord of a 6-month-old tau Tg mouseshows axonal inclusions that are immunolabeled with monoclonal anti-human tau antibody T14. (B) High-power view of the tau immuno-reactive spheroid indicated by the arrow in A. (C) The spheroidal inclusion (arrow) is immunolabeled with polyclonal antibodies againstalpha-tubulin. (D–F) Triple immunofluorescence stain demonstrates that the T14-positive inclusion (D) is also stained with polyclonalanti-NFL antibodies (E) and monoclonal anti-NFH antibody DP1 (F). (G) Transmission electron microscope (EM) image shows that theintra-axonal inclusion (arrow) consists of randomly oriented 10- to 20-nm straight filaments. Arrowhead indicates a degenerating organelle.The section was generated using spinal cord of a 6-month-old tau Tg mouse. (H) High-power view of the filamentous inclusion indicated bythe arrow in G. Mitochondria is contained at the center. (I) The inclusions are shown to contain tau filaments by postembedding immuno-EMwith the anti-tau antibody T14. Note that the area outside the inclusion does not show any immunolabeling. (J) Immunoblots showprogressive accumulation of insoluble tau proteins in the brain cortex (Cx) and spinal cord (SC) of the tau Tg mice. The protein samples withdifferent solubilities were extracted using RAB Hi-Salt (RAB-HS), RIPA, and 70% formic acid (FA), in order of insolubility. The insoluble

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and spinal cord (20; Fig. 2). The spinal cords and brainsfrom 1-, 3-, 6-, and 9-month-old line 7 Tg and WT micewere sequentially extracted with buffers of increasingextraction strength, i.e. reassembly buffer (RAB),RIPA buffer, and 70% formic acid (FA). The three frac-tions were then analyzed by quantitative Western blot-ting with antibody 17026. In the WT mice, about 90%of endogenous mouse tau in both the brain and thespinal cord was RAB soluble and no immunoreactivitywas detected in the FA-soluble fraction. In the Tg mice,although the quantity of RAB-soluble tau remainedconstant at around 75–80% with increasing age, thequantity of RAB-insoluble tau represented by the RIPAand FA fractions progressively accumulated in boththe brain and the spinal cord. For example, RIPA-soluble tau in brain increased from about 17% at 1month to about 25% at 9 months and FA-soluble tau inspinal cord increased from about 0.4% to about 1.8%over the same time period. The accumulated RAB-insoluble tau was mainly Tg tau, and this accumula-tion correlated with the emergence of tau pathology inthe Tg mice. In addition, RAB-insoluble tau, especiallythe FA fraction, was more pronounced in spinal cordthat in brain, consistent with more abundant tau ag-gregates in the spinal cord.

The Phosphorylation State of Tau in the Tau Tg MiceRecapitulates That in ALS/PDC

Western blot analyses of soluble and insoluble tauextracted from the cerebral cortex of Tg mice wereperformed using the phosphorylation-independent an-tibody 17026, which recognizes all tau forms. (20). Sim-ilarly, T14 recognizes all but the mouse tau sample.MAb T1 (specific for a tau epitope that is not phosphor-ylated at amino acids 189–207) did not recognize PHF-tau, but was immunoreactive with human adult nor-mal tau, fetal tau, and both soluble and insoluble Tgtau. This indicates that tau from the Tg mice is par-tially dephosphorylated at the T1 epitope. However,several phophorylation-dependent antibodies, whichreact with PHF-tau and fetal tau, but not with normaladult tau, also recognized both soluble and insolubletau from the Tg mice. These antibodies include PHF1(phosphoserines 396 and 404), T3P (phosphoserine396), PHF6 (phosphothreonine 231), AT8 (phospho-serines 202 and 205), AT270 (phosphothreonine 181),and 12E8 (phosphoserine 262). Therefore the phos-phorylation state of Tg tau recapitulates that of PHF-tau found in human tauopathies including ALS/PDC(1, 3, 22, 26).

Tau Tg Mice Develop Gliosis, Axon Degeneration, andReduced Fast Axonal Transport Linked toProgressive Motor Weakness

To detect astrocytosis, a MAb to glial fibrillary acidicprotein was used, and it stained numerous reactiveastrocytes in brain and spinal cord of Tg, but not WT,mice, indicating the presence of profound gliosis inregions with neuronal damage (20). Further, the astro-cytosis was age dependent, corresponding to the devel-opment of the tau inclusions. Since inclusions in theproximal axons of affected neurons could cause diseaseby damaging axons, we examined the morphology ofspinal cord ventral root axons. In semithin sections,the normal L5 ventral root of a WT mouse containedmany large- and small-sized myelinated axons, but theventral root of a 6-month-old Tg mouse contained pri-marily irregularly shaped axons. By 12 months of age,the endoneurial space appeared to increase, consistentwith the removal of degenerated axons in these nerves.Evidence of axonal degeneration also came from com-paring axon numbers in L5 ventral roots of Tg and WTmice. Although at 6 months of age, the number of axonsin the Tg mice was comparable to that of age-matchedWT mice, a 20% decrease was seen in 12-month-old Tgmice. Also, despite a significant reduction of MT den-sity in the 12-month-old Tg mice, the NF density re-mained unchanged compared with age-matched WTmice. This finding correlated with the biochemicalanalysis of alpha-tubulin and NF subunits in the prox-imal sciatic nerve, which showed a progressive de-crease in alpha-tubulin level in the Tg mice and rela-tively constant levels of NF subunits.

To assess whether axonal transport was compro-mised in the Tg mice, radiolabeled proteins trans-ported in the fast component were measured followingmiroinjection of [35S]methionine into the L5 ventralhorn of 12-month-old tau Tg and age-matched WT mice(20). This showed retarded fast axonal transport ofradiolabeled proteins in the tau TG mice. The largestdifferences in the amounts of transported radiolabeledproteins appeared around the 4-mm segment wherealmost twice as many radiolabeled proteins were de-tected in WT compared to tau TG mice. Finally, inaddition to acquiring the spinal cord pathologies de-scribed above, the tau Tg mice also developed progres-sive motor weakness, as demonstrated by their im-paired ability to stand on a slanted surface and byretraction of their hind limbs when lifted by their tails.This is similar to the clinical and neuropathological

human transgenic tau (FA fraction) accumulates in the spinal cord more rapidly than in the cortex (left panels). Although the endogenousmouse tau in Tg mice also shows slight increase in insolubility with aging, the mouse tau is much less insoluble than human tau (compareleft and middle panels). The endogenous mouse tau in either cortex or spinal cord of WT mice does not change in insolubility with aging (rightpanels). The antibodies against human tau (T14) and mouse tau (T49) are used here. Scale bars, 100 �m (A and C), 20 �m (B), 50 �m (D–F),1000 nm (G), 250 nm (H), 500 nm (I).

7MODELS OF NEURODEGENERATIVE TAUOPATHIES OF CHAMORROS

phenotype observed in other lines of tau transgenicmice (9, 18, 34).

Old Tau Tg Mice Develop Congophilic Fibrillary TauInclusions and Their Tauopathy Is Attenuated inTau Tg Mice That Lack NFs

In subsequent studies designed to assess the effect ofold age on the properties of the intraneuronal filamen-tous tau inclusions described above, 12- to 24-month-old Tg mice overexpressing the fetal tau isoform wereexamined (21). With advancing age beyond 12 months,these tau Tg mice progressively developed more abun-dant tau inclusions that were frequently congophilic inseveral brain regions including the hippocampus,amygdala, and entorhinal cortex. These congophilicNFT-like inclusions were first detected in Tg mice at18–20 months of age and they were demonstrated byhistochemical dyes that bind specifically to crossed�-pleated sheet structures (e.g., Congo red and Thio-flavin S). Ultrastructurally these tau inclusions con-tained straight tau filaments composed of both mouseand human tau, like their counterparts in the youngerTg mice, but they did not contain other cytoskeletalproteins such as the NF and tubulin proteins seen inthe inclusions of younger mice. Furthermore, isolatedtau filaments could be recovered from detergent-insol-uble tau fractions in the brains of the older Tg mice,and insoluble tau proteins accumulated in their brainsin an age-dependent manner. Thus, these studies showthat overexpression of a single tau isoform resulted inthe late-onset, age-dependent formation of congophilictau inclusions with properties similar to those of tautangles observed in Guam ALS/PDC and other relatedhuman tauopathies, thereby implicating aging in thepathogenesis of fibrous tau inclusions.

Finally, in view of the fact that NF proteins werepresent both in the tau inclusions of the young tau Tgmice (20) and in the tau inclusions in human tauopa-thies (20, 25, 29, 37, 41), we sought to elucidate the roleof NF subunit proteins in tau aggregate formation totest the hypothesis that NFs might function as “patho-logical chaperones” in the formation of intraneuronaltau inclusions (19). To this end, we crossbred the pre-viously described tau (T44) Tg mice overexpressing thesmallest human tau isoform with knock-out mice de-void of NFL (NFL�/�) or NFH (NFH�/�). Notably,depletion of NF subunit proteins from the T44 mice(i.e., T44;NFL�/� and T44;NFH�/�), and especiallyNFL, resulted in a dramatic decrease in the total num-ber of tau-positive spheroids in spinal cord and brainstem. Concomitant with the reduction in spheroidnumber, these bigenic mice showed delayed accumula-tion of insoluble tau proteins in the CNS, increasedviability, reduced weight loss, and amelioration of theirmotor impairment compared to the single T44 Tg mice.Thus, these results imply that NFs are “pathological

chaperones” involved in the development of tau sphe-roids and suggest that NFs play a role in the patho-genesis of neurofibrillary tau lesions in Guam ALS/PDC and in other neurodegenerative tauopathies.

DISCUSSION

As summarized here, there is now compelling evi-dence from our studies and those from other groups (9,18–21, 23, 34,) that the overexpression of tau proteinsin Tg mice causes a CNS neurodegenerative tauopathywhich recapitulates key aspects of human tauopathiessuch as Guam ALS/PDC, PSP, and some FTDP-17syndromes. For example, in our tau Tg mice, we ob-served a progressive, age-dependent accumulation ofargyrophilic tau immunoreactive inclusions in neuronsof spinal cord, brainstem, and neocortex similar tohuman tauopathies (18–21). Since the inclusions in theTg mice were most abundant in spinal cord neurons,the tauopathy in these mice most closely resemblesALS/PDC wherein tangles are abundant in spinal cord(25, 35, 40). Significantly, ALS/PDC patients whopresent with motor weaknesses do so about a decadeearlier than those who present with parkinsonism anddementia. Thus, the accumulation of tau aggregates inthe brains of our Tg mice later than in spinal cordmirrors disease progression in ALS/PDC patients whopresent with motor weakness. Moreover, as discussedhere, these tau tangles also are immunostained byantibodies to NF proteins and tubulin, as are the in-clusions in our Tg mice. Finally, ALS/PDC is associatedwith a progressive motor weakness similar to that ob-served in the Tg mice.

Although the distribution of the tau pathology inthese mice most closely resembles that found in ALS/PDC, PSP, CBD, and some FTDP-17 syndromes, thesefilamentous tau aggregates share many characteristicswith authentic NFTs in AD and other tauopathies.First, like highly insoluble PHF-tau in AD NFTs, asubstantial fraction of tau proteins from the Tg mice isextracted only with RIPA and FA despite the fact thatnormal tau is highly soluble in aqueous solutions (20,21). Second, the amount of insoluble tau protein pro-gressively accumulates with age and disease progres-sion in the Tg mice similar to ALS/PDC, AD and othertauopathies. Third, PHF-tau in human NFTs are hy-perphosphorylated and so are the soluble and insolublespecies of tau recovered from Tg mice. Fourth, al-though ALS/PDC and AD NFTs contain mostly PHFs,straight filaments similar to those found in the inclu-sions of the Tg mice are also present. Finally, in addi-tion to ALS/PDC, NF protein immunoreactivity alsooccurs in NFTs of AD, PSP, and other tauopathies.

Despite similarities between the tau aggregates inour Tg mice and those of other tauopathies, the tauinclusions in the Tg mice are not identical to AD andALS/PDC NFTs. For example, ALS/PDC and AD NFTs

8 TROJANOWSKI ET AL.

contain all six tau isoforms, while the tau pathology inTg mice engineered to overexpress the smallest humantau isoform does not. In this regard, the filamentous Tgtau inclusions in these Tg mice resemble Pick bodies inthe brains of patients with typical Pick’s disease sincethese pathological bodies are comprised mainly of tauisoforms with three MT binding repeats (3R-tau) inmost, but not all, cases of Pick’s disease (47). Onenotable difference, however, is that all three brain 3R-tau isoforms are found in Pick bodies, whereas fetal tauis the only 3R-tau isoform present in the tau aggre-gates of our Tg mice. Thus, insoluble tau tangles arecomposed of all six tau isoforms in AD and ALS/PDC,or predominantly the three isoforms with four MT (4R-tau) binding repeats in PSP and CBD, or mostly 3R-tauin Pick’s disease. Further, recent studies of FTDP-17and other familial tauopathies suggest that tau tanglescomposed of different ratios of the six alternativelyspliced tau isoforms exist in different tauopathies (6,23, 46). However, further studies are needed to eluci-date how the accumulation of specific subsets of tauisoforms is linked to the degeneration of different celltypes and the diverse clinical manifestations of dis-ease.

Since the accumulation of filamentous tau inclusionsin spinal cord neurons was associated with the degen-eration of ventral root axons in the tau Tg mice, wehypothesize that this reflects a gain of toxic function bythe overexpressed tau, and several lines of evidencesupport this hypothesis. First, previously described tauTg mice that expressed lower levels (� twofold) of tauprotein did not develop filamentous tau inclusions orneurodegeneration. Second, we observed a dose-depen-dent increase in the size and number of tau aggregatesin out two lines of Tg mice. Thus, one plausible expla-nation to account for the axonal degeneration in theseTg mice is a gain of toxic function by the excess tauproteins that cannot bind MTs because all of the MTbinding sites are occupied, which leads to the aggrega-tion and fibrillization of this excess, unbound tau inneurons followed by a blockage of perikaryal and ax-onal transport and the degeneration of affected axons.

The reduced numbers of MTs and the reduced levelsof tubulin, but not NFs or NF proteins, in the remain-ing axons of the degenerating ventral roots also implya loss of the MT-stabilizing function of tau. Since over-expressed human tau aggregated with endogenousmouse tau leading to progressive insolubility and hy-perphosphorylation of both human and mouse tau inthe Tg mice, this could impede endogenous mouse taufrom performing its MT-stabilizing function. Indeed,the observed reduction in fast axonal transport in 12-month-old Tg mice is consistent with a loss of MTfunction, although the loss of axons in the Tg mice maycontribute to this. Based on indirect evidence fromstudies of human tauopathies, we and others haveproposed that both gains of toxic functions and losses of

normal tau functions could be involved mechanisticallyin causing neurodegenerative disease (6, 17, 23, 46),and the data presented here support these mechanistichypotheses.

Although a dose effect of the transgene is observed intwo different Tg mouse lines, the distribution of thetau-rich lesions within the CNS is not completely de-pendent upon the expression levels across the differentregions. For example, spinal cord expresses less Tg tauthan brian, but more abundant and larger inclusionsaccumulate earlier in spinal cord neurons than in theother brain regions. This could be explained by meta-bolic differences among diverse types of neurons or bythe aggregation of excess tau at lower concentrationsin spinal cord versus other neurons under the influenceof local factors or “pathological chaperones” such ashigh concentrations of NF proteins. Similarly, the se-lective distribution of tau pathology in different humantauopathies is likely due to other as yet unidentifiedlocal vulnerability factors. Indeed, the findings de-scribed here parallel the well-known, but enigmatic“selective vulnerability” that is a constant feature ofmost human neurodegenerative diseases. In addition,our studies of the bigenic mice generated by crossingthe tau Tg mice with NF knock-out mice supports thehypothesis that NF proteins may function as “patho-logical chaperones” in disease pathogenesis becausethe phenotype was attenuated relative to the tau Tgmice with a normal complement of NFs. Since the Tgtau mice described here exhibit the key neuropatholog-ical features of Guam ALS/PDC, they will be useful instudies designed to further elucidate mechanisms lead-ing to the formation of tau pathology and the selectivedegeneration of neurons in ALS/PDC and relatedtauopathies including AD. Indeed, based on very prom-ising results from studies of new potential AD thera-pies in Tg mice that model AD amyloidosis, it is plau-sible to anticipate similar developments in efforts totreat tauopathies based on the use of compounds thateither counteract the loss of tau when it becomes se-questered in tangles, thereby impairing axonal trans-port, or disrupt the fibrillization and aggregation of tauinto filamentous inclusions (22, 42).

ACKNOWLEDGMENTS

We thank Drs. M. K. Lee and M. L. Schmidt and all of the membersof the Guam Project for collaborations in our studies of ALS/PDC andthe generation of the tau Tg mice. We also thank N. Shah, E.Heatherby, K. H. Szymczyk, and G. Kim for technical assistance. Weexpress our deep appreciation to the Chamorro community on Guamand especially to the Chamorro patients and their families, whosegenerous support for the Guam Project made our research possible.V. M.-Y. Lee is the John H. Ware 3rd Professor of Alzheimer’sDisease Research at the University of Pennsylvania, and the studiesreviewed here were supported by grants from the NIA. This review isdedicated to the memory of Dr. Wigbert C. Wiederholt, the formerChair of Neuroscience at University of California at San Diego, whosuccessfully led the effort to reestablish a multidisciplinary NIH-

9MODELS OF NEURODEGENERATIVE TAUOPATHIES OF CHAMORROS

funded Guam Project to pursue research on Chamorro neurodegen-erative diseases.

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