Vikram Khurana

M.D Mel B Feany
email: mel_feany@hms.harvard.edu

Connecting cell-cycle activation to neurodegeneration in Drosophila

Studies in cell-culture systems and in postmortem tissue from human disease have suggested a connection between cell-cycle activation and neurodegeneration. The fruit fly Drosophila melanogaster has recently emerged as a powerful model system in which to model neurodegenerative diseases. Here we review work in the fly that has begun to address some of the important questions regarding the relationship between cell-cycle activation and neurodegeneration in vivo, including recent data implicating cell-cycle activation as a downstream effector of tau-induced neurodegeneration. We suggest how methods in the fly might be utilized to approach fundamental questions that remain.

Introduction

In 1992, a report by Feddersen, Orr and colleagues showed that SV40-mediated disruption of the tumor suppressor pRb leads to Purkinje cell degeneration [1]. In an insightful perspective piece that followed this study, Nathaniel Heintz postulated that there may be a fundamental relationship between neurodegeneration and neoplastic transformation and that "genes that can cause transformation in dividing cell populations (and thus result in clonal expansion and neoplastic disease) can cause programmed cell death of terminally differentiated neurons (and thus result in clonal elimination and neurodegenerative disease)" ( [2]; Figure 1). In the last decade, an important body of work in cell-culture systems and in postmortem tissue from human disease has further raised the possibility of a mechanistic connection between transformation and neurodegeneration [3]. There have, for example, been several studies indicating that posmitotic neurons abnormally re-express numerous cell-cycle markers and replicate their DNA in neurodegenerative disorders, including tauopathies and Alzheimer's disease (AD) [4,5]. In

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3 various neurotoxic paradigms in cell-culture systems, neuronal apoptosis can be prevented by blocking various components of the cell-cycle machinery [3]. Finally, certain cell-cycle regulators, including E2F and Cdc2, have been shown to directly activate the neuronal apoptotic machinery [6][7][8][9].

For all their strengths, studies associating aberrant cell-cycle marker expression with neurodegeneration in postmortem tissue have been largely correlative. For example, immunostaining in these studies has often only been in abnormal structures such as neurofibrillary tangles (NFTs), proteinaceous aggregates of the microtubule-associated protein tau that could potentially bind antibodies non-specifically. Also, proteins expected to be nuclear during cell-cycle progression have sometimes been found to be cytoplasmic in dying neurons [10,11]. Thirdly, both cell-cycle activating and inhibitory proteins are upregulated in AD and animal models [11,12], making it difficult to conclude whether activation or inhibition of the cell cycle, or neither, contribute to neuronal demise. These findings raise the concerns that either aberrant cell-cycle marker expression indicates nothing more than dysregulated protein synthesis in dying neurons or, alternatively, that the upregulated proteins may be subserving non cell-cycle roles. In this regard, Herrup and colleagues made a significant advance by showing that DNA is actually replicated in susceptible and dying neuronal populations in AD, supporting the idea that neurons re-enter a co-ordinated cell cycle in this disease. However, to add complexity, DNA replication and abnormal cell-cycle marker re-expression in postmitotic neurons has been described in several animal models in which neurons do not appreciably degenerate, including rodent models of ATM [13] and AD [14], and in Retinoblastoma Protein-1 (pRb-1)-deficient mice. For example, while widespread neurodegeneration occurs in the central nervous system (CNS) of pRb-1-deficient mice [15,16], this effect is non cell-autonomous because in chimeric

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4 mice, the pRb-1 -/-clones in the CNS exhibit tetraploidy in the absence of appreciable neuronal death [17].

Bearing these data in mind, we wondered first whether there was a causal relationship between cell-cycle activation and neurodegeneration in animal models of tauopathy and second, whether cell-cycle activation was sufficient to cause neurodegeneration. Third, if cell-cycle activation did mediate neuronal death, it remained to be seen which phase of the cell cycle was lethal for a neuron. The absence of any description of mitotic figures or cytokinesis of a postmitotic neuron certainly implied that adult neurons could not progress through mitosis, although studies in cell-culture systems also implicated early G1/S mediators, such as E2F and Cyclin D, in causing neuronal apoptosis. Finally, the question of the triggers for postmitotic neuron to re-enter the cell cycle remained unknown. In particular, in postmortem tissue from AD, numerous mitogenic signaling molecules had been shown to be upregulated [18][19][20][21][22], but the consequences of these pathways being activated in postmitotic neurons, whether apoptotic or pro-mitotic, were unclear.

In this context, we felt the genetically tractable fruit fly, in which neurodegenerative diseases are now modeled, had a unique role to play. In this review, we highlight the utility of this model organism to investigate the connection between cell-cycle activation and neurodegeneration. We analyze recent experimental data that have begun to address some of the important questions in the field, and discuss how methods in the fly might be utilized to approach fundamental questions that remain.

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The utility of Drosophila in investigating the cell-cycle/neurodegeneration connection

Drosophila is ideally suited to investigate the relationship between neurodegeneration and cellcycle activation in vivo for several reasons. First, the cell-cycle machinery is substantially conserved between Drosophila and mammalian systems [23]. Indeed, considerable advances in our understanding of the role of cell-cycle mediators in vivo have been afforded by investigations in flies. Second, there are now several well-established Drosophila models of neurodegeneration, including tauopathies, Parkinson's disease (PD) and polyglutamine disorders (reviewed in [24]).

As noted above, postmortem analyses associate cell-cycle activation with tauopathies including AD. Of primary importance, the relative low level of redundancy in the fly genome (that is, a single gene in Drosophila is often the only homolog for an entire family of mammalian genes)

and the availability of many sophisticated genetic tools make genetic analysis in Drosophila well suited to address issues of causality. Finally, the short lifespan and reproductive cycle of flies allow for the analysis of many potential genetic modifiers in a relatively short amount of time.

Cell-cycle and mitogenic signaling machineries are well conserved in Drosophila

In the cell cycle, DNA synthesis occurs during S-phase and mitosis occurs in M-phase. Two gap phases (G1 and G2) separate the S-and M-phases. Between division cycles, cells are in G0.

Neurons are generally considered postmitotic and permanently in G0 (Figure 2).

Immunohistochemical markers can be used to identify cell-cycle phases. For example, the proliferating cell nuclear antigen (PCNA), thought to function as a "sliding clamp" for DNA polymerase-δ [25], is upregulated during G1, S and G2. Phosphorylation at Ser10 of histone-3 (PH3) occurs during condensation of chromosomes in M-phase [26] and is thus considered an M-phase-specific marker. It should be noted, however, that these markers are upregulated in

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situations other than cell division -for example, PCNA is involved in DNA repair [27] and H3 phosphorylation at Ser10 occurs during immediate-early gene transcription [28]. 5-bromo-2'deoxyuridine (BrdU) incorporation into actively synthesized DNA is a more definitive S-phase marker.

Cyclin-dependent kinases (Cdks) catalyze reactions essential for progression through the cell cycle. Key regulators of the G1/S checkpoint include the E2F family of transcription factors, the pocket proteins (including pRb) and the Cdk inhibitors p21 and p27 (Figure 2). E2F factors 1 and 2 can directly activate the transcription of many S-phase genes, including DNA polymeraseα and Cyclin E [29]. Pocket proteins of the pRb family can directly bind and inhibit E2Fmediated transactivation [30]. This inhibition is relieved by pocket protein phosphorylation by Cdks. Alternatively, E2F 4 and 5 family members can bind pocket proteins and form complexes that repress transcription. p21 and p27 regulate the G1/S transition by directly binding and inhibiting Cdk2/Cyclin E. These inhibitors can also inhibit DNA synthesis by binding PCNA [31].

In Drosophila, the essential features of the cell cycle are conserved [23]. Homologs of Cdks, Cyclins, Cip inhibitors, E2F transcription factors and pocket proteins have been described [32]. Drosophila homologs are indicated in Figure 2 (black). Cdk1 and Cdk2 are often referred to in the literature as cdc2 and cdc2c, respectively. Note that Drosophila Cyclin A complexes with Cdk1 but does not complex with Cdk2. Cyclin E, however, does complex with Cdk2 to facilitate the G1/S transition. The G2/M transition is dependent upon Cdk1/Cyclin A in flies, with Cdk1/Cyclin B complexes thought to be important in progression through the various stages of mitosis. The fly homolog for p27/p21 is Dacapo (Dap) [33]. There are two E2f family members in Drosophila [34]. E2f1 directly transactivates gene transcription, like the vertebrate

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E2Fs 1/2/3, and is inhibited by Rbf, the homolog of pRb-1. E2f2, like the vertebrate E2Fs 4/5, forms repressor complexes with Rbf2. In studies of Drosophila development, inhibition of the G1/S transition is achieved by overexpressing Rbf1, dE2f2, Rbf2, Dacapo or human p21 [33,34]. The G1/S transition appears to be most effectively inhibited in the developing larval eye disc by co-expressing both Dap and Rbf1 [33].

The primary function of Cdk4/Cyclin D during fly development appears to be as a growth activator, although Cdk4/Cyclin D drives the G1/S transition indirectly in some tissues [35,36].

Similarly, classic mitogenic and oncogenic signaling pathways, including Myc [37], Ras [38] and TOR [39], are highly conserved in flies and appear to drive the G1/S transition indirectly by promoting cellular growth. Interestingly, several papers have shown that TOR activity is abnormally upregulated in AD brain [18,40].

A Drosophila model of tauopathy

In postmortem studies, early cell-cycle markers including PCNA are upregulated in a number of acute and chronic neurodegenerative conditions [6]. In contrast, markers of the G2/M transition appear to be more selectively upregulated in tauopathies [4]. Tauopathies are neurodegenerative diseases characterized by intraneuronal accumulations of hyperphosphorylated forms of the microtubule-associated protein tau. The term encompasses hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and sporadic tauopathies including AD, progressive supranuclear palsy and Pick's disease. We, and others, have developed Drosophila models of tauopathies [41,42]. Transgenic flies are created by expressing human wild-type or FTDP-17-associated mutant tau constructs. Tissue-specific expression of the transgenes is achieved using the bipartite UAS/GAL4 system derived from yeast [43]. For example, GMR-

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GAL4 drives expression within retinal tissue, in both neuronal (photoreceptors) and non-neuronal postmitotic cells. elav-GAL4 drives transgene expression within postmitotic neurons, including photoreceptors of the retina and the brain (Figure 3H).

Tau expression induces retinal and brain phenotypes that have been well characterized in our laboratory and by other investigators [41,42,44]. Retinal toxicity is observed in adult flies as a "rough" eye phenotype. The eye is reduced in size, loses its regular ommatidial structure and is irregularly contoured (Figures 3A-D). The rough eye phenotype is useful because it is easily scored and amenable to genetic modification, making it a valuable tool for both forward (unbiased) and candidate genetic screens. Recently, this phenotype was used to conduct a P element-based forward modifier screen for tau-induced neurotoxicity [45].

When tau is expressed with elav-GAL4, adult flies eclose with morphologically normal brains. However, in adult life, accumulation of disease-associated phospho-and conformational tau epitopes accompanies progressive neurodegeneration, histologically characterized by neuronal nuclear fragmentation and vacuolization of the cortex and neuropil (Figures 3 F-G) [42,44], and TUNEL (terminal transferase-mediated deoxy-UTP biotin nick end-labeling) staining of apoptotic neurons.

Investigating cell-cycle activation in a Drosophila tauopathy model

Establishing a causal connection between tau-induced neurodegeneration and cell-cycle activation in vivo

Recently, we found that, as in tauopathies, the cell-cycle markers PCNA and PH3 were abnormally expressed in neurons in the Drosophila tauopathy model [46]. These could always be

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found in transgneic tau fly brains, but were completely absent in age-matched control flies.

PCNA staining was both cytoplasmic and nuclear, and PH3 staining was entirely nuclear, consistent with the subcellular localization patterns during cell-cycle activation. In agreement with previous postmortem and cell-culture studies, we did not see abnormal cytokinesis of postmitotic neurons in the brains of transgenic tau flies.

A comprehensive genetic analysis in the retina and brain (Table 1) established a clear causal relationship between cell-cycle activation and neurodegeneration. In other words, the use of mutant alleles or transgene combination to inhibit cell-cycle progression substantially reduced tau-induced neuronal apoptosis. In contrast, co-expressing genetic activators of the cell-cycle synergistically enhanced tau-induced neurodegeneration. Cdk1 and Cdk2 enhanced tau-induced neurodegeneration when co-expressed with their respective Cyclins. Since multiple reagents at both the G1/S and G2/M transition fitted this pattern, our data strongly implicated the cell-cycle pathway as a whole in mediating tau-induced neurodegeneration. There were rare exceptions to the rule. For example, two separate Cyclin E mutants did not suppress tau-induced retinal degeneration. This may be trivially related to haplosufficiency of Cyclin E. Notably, however, these alleles do exert dominant effects in developmental contexts [47] and, overall, the effects of modulating the G2/M Cyclins A and B were certainly more striking in modifying tau-induced toxicity than the effects of modulating the G1/S Cyclins D and E.

Postmortem analyses of AD and tauopathy tissue have associated cell-cycle marker expression with abnormal tau phosphorylation. Since Cdks are well known to phosphorylate tau [48], the question of whether cell-cycle activation acts up-or downstream of tau phosphorylation Since cell-cycle activation occurred downstream of abnormal tau phosphorylation in the fly tauopathy model, cell-cycle activation might directly precede apoptosis [46]. We tested whether ectopic cell-cycle activation was sufficient to trigger apoptosis by using elav-GAL4 (or the inducible GeneSwitch-elav) drivers to express E2f1/Dp, Cdk2/Cyclin E and Cyclin A in neurons. We found that increased TUNEL staining accompanied upregulation of PCNA and PH3, indicating that ectopic cell-cycle activation is sufficient to trigger neuronal apoptosis.

Ectopic E2f1/Dp expression, in particular, lead to dramatic neuronal apoptosis and cell loss commensurate with the degree of cell-cycle marker upregulation. Diseases. It should be noted, however, that these experiments do not rule out potential cell-cycle-independent apoptotic effects of ectopic cell-cycle activation. For example, E2f1/Dp is known to directly transactivate proapoptotic genes in cell-culture system and in flies [49]. Interestingly, our preliminary data

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indicate that cyclins vary in their efficacy to induce apoptosis, and that different neuronal populations within the brain respond differently to the same cell-cycle stimulus.

Determining the link between tau and cell-cycle re-entry

Having established a causal relationship between cell-cycle activators and tau-induced neurodegeneration, we focused on the mechanism of tau-induced cell-cycle activation. Potential mechanisms include those more closely related to tau, both in terms of its role as a microtubuleassociated protein and its amino acid sequence, and general mechanisms, including oxidative stress, DNA damage and inhibition of the ubiquitin/proteasome system (Figure 4). The general mechanisms each have precedents in cell-culture systems. Our data indicated that ectopic cellcycle marker expression was not a feature of the PD and spinocerebellar ataxia-3 (SCA3) fly models, and cell-cycle modification did not modify the SCA3-induced retinal p henotypes, supporting the possibility that cell-cycle activation may be tau-specific [46].

Intriguing data from cell-culture systems implicate tau as a potential signaling molecule.

Tau has been identified at the cell membrane and is enriched in axon growth cones. Furthermore, transfection of tau into neuronal cell lines results in neurite extension, suggesting that tau is capable of activating complex and coordinated intracellular signals [50,51]. Tau is well known to be phosphorylated at many disease-associated phosphoepitopes in fetal life [52], a period of dynamic growth cone changes, and tau-induced neuritic extension in cell culture studies is modulated by Ser-Pro/Thr-Pro phosphorylation [50]. Phosphorylated tau may thus be particularly effective at transducing growth signals, a finding that may have relevance for disease states in which tau is hyperphosphorylated. As noted above, tau phosphorylation was required for tau-induced cell-cycle activation in the fly tauopathy model.

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The amino acid sequence of tau reveals 5 tyrosines, 7 N-terminal PXXP motifs and multiple SP/TP motifs (Figure 5). Since SH2 domains bind phosphotyrosine, SH3 domains bind proline-rich domains that commonly contain a "PXXP" motif and WW domains bind SP/TP motifs, tau could potentially bind proteins containing these modular domains and be capable of Hyperactive RTK signals can lead to oncogenesis through multiple mechanisms including increased transcription and translation of necessary components for growth and proliferation [53]. Viral antigens, such as the large and small SV40 T antigens, are other examples of proteins involved in multiple interactions, including with pRb, protein phosphatase 2A (PP2A) and p53, that induce cellular transformation [54]. In fact, the literature reveals that tau also binds several SH2/SH3 and WW domain-containing proteins, including non-receptor tyrosine kinases of the Src-family and c-Abl, the prolyl isomerase Pin-1 and phospholipase C-γ (PLC-γ). An intriguing association has recently been described between the N-terminal of tau and the pRb-binding protein Che-1 [55]. Che-1 blocks both pRb-1 function in human cell lines by competing for HDAC binding [56]. The direct binding between tau and a negative regulator of the cell cycle is of obvious interest in the context of investigating the connection of tau to the cell cycle.

Interestingly, tau is a substrate of the neuronally enriched PP2A and PP2A activity is decreased in AD [57][58][START_REF] Sontag | 3rd[END_REF][60].

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Since fly homologs for classic oncogenic signaling pathways and known tau interactors exist, we tested many signaling molecules for genetic modification of the tau-induced rough eye.

Our candidate analysis is ongoing, but has hitherto strongly implicated signaling pathways known to activate growth as mediators of tau-induced neurotoxicity. Since the TOR pathway is a point of convergence for multiple growth-activating pathways (Figure 6; [61]) and TOR signaling has been linked to aging [62,63] and AD, we thoroughly investigated the effect of modulating this pathway on tau-induced neurodegeneration [46]. We consistently found that TOR-activated genetic backgrounds synergistically enhanced tau-induced neurotoxocity, and TOR inhibition ameliorated tau-induced neurodegeneration in the retina and brain (Table 2). Our data indicated that TOR-dependent enhancement of tau-induced neurodegeneration could be blocked by concomitant cell-cycle inhibition and that ectopic TOR activation induced aberrant neuronal cell-cycle marker expression and apoptosis. These data strongly implicate TOR as an important mediator of tau-induced cell-cycle activation, although do not rule out parallel pathways existing for tau-induced cell-cycle activation.

Fundamental questions that remain and how Drosophila might help

The Drosophila tauopathy model has proved useful in resolving three issues to date. factors can directly transactivate pro-apoptotic genes [9], and that Cdk1 can directly lead to BAD-dependent neuronal apoptosis [8]. In contrast, postmortem tissue analyses in AD and AD mouse models suggest that DNA has been replicated in susceptible postmitotic neurons.

Furthermore, in cell-culture AD models, neuronal apoptosis can be blocked by inhibiting DNA polymerase [3]. These latter investigations would support a coordinated cell-cycle progression being potentially responsible for neurodegeneration. The distinction is biologically important

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15 because, in the former case, cell-cycle activators are simply upstream components of the neuronal apoptosis mechanism. In the latter case, cell-cycle processes, the replication of DNA or the G2/M transition for example, may be responsible for cell death.

The Drosophila tauopathy model may be useful in resolving this issue, although more definitive measures of cell-cycle phasing are required. Such techniques would include assays for neuronal BrdU incorporation and FISH or fluorescence-activated cell sorting (FACS) to directly assess neuronal ploidy. BrdU incorporation [33,64] and FACS [35] are standard techniques that have been used to determine the effect of cell-cycle modulators in proliferating fly larval tissues.

We have not yet observed BrdU incorporation in the fly brain of transgenic tau flies, although whether this is a true negative result or simply reflects the technical limitations of the experiment remains unclear. It is important to note that relatively few neurons in the brain of transgenic tau flies demonstrate re-expression of cell-cycle markers at any given time, and thus negative results from any of these approaches would be difficult to interpret. An alternative approach would be to express tau in proliferating tissue, such as fly imaginal discs in which ploidy analysis is a wellestablished technique. Coupled with FACS analysis, this may provide interesting insights into how tau affects cell-cycle phasing. Our preliminary data would suggest that tau expression in imaginal discs does in fact lead to marked increases in cell-cycle marker activation, indicating either hyperproliferation or delayed cell-cycle exit.

Is neuronal cell-cycle activation sufficient to cause neuronal apoptosis?

The question of sufficiency of cell-cycle activation for neuronal apoptosis has plagued the cellcycle/neurodegeneration field since its inception with the observation that DNA replication can be observed in the absence of neurodegeneration in a variety of context in postmitotic neurons,

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16 including in Rb-/-chimeric mice, Atm-/-mice, APP transgenic mice and in certain populations of neurons in AD brain [5,13,14,17]. Our data would indicate the ectopic expression of multiple cyclins and E2f/Dp in the brain is sufficient to drive apoptosis of postmitotic neurons in vivo [46]. Interpretation of these experiments, however, is complicated by PCNA and PH3 being our Investigating the issue of sufficiency is important for several reasons. First, understanding the phase at which the apoptotic signal is transduced may have significant implications for designing appropriate therapies. Also, events occurring up-or downstream of tau-induced cell-cycle activation may be critical for an apoptotic response once the cell cycle becomes activated [65]. These events could also potentially be therapeutic targets. Recently, it has been proposed that oxidative stress may be one such critical upstream event [65,66]. For example, a neuron might be able to enter an S-phase without detriment, unless its DNA has previously been damaged. Thus, an attempt to replicate damaged DNA may be the trigger for apoptosis rather than cell-cycle re-entry itself [67], and oxidative stress may be one important

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cause of such DNA damage. We have recently showed that genetic and pharmacologic manipulation of oxidative stress modifies tau-induced neurodegeneration in flies (Dias et al., in press), and thus the fly tauopathy model may be very useful in investigating the cellcycle/oxidative stress connection. Determining critical upstream events may also shed light on the tau-induced phenotype itself because, while ectopic TOR and cell-cycle activation can lead to neuronal apoptosis in the fly brain, in neither situation is the tau phenotype precisely recapitulated, suggesting that events occurring upstream of TOR/cell cycle may be crucial determinants of this phenotype.

Are there critical non cell-autonomous factors that mediate cell-cycle-dependent apoptosis?

An issue related to sufficiency of cell-cycle activation for neuronal apoptosis is discerning cellautonomous from non cell-autonomous factors in the mediation of cell-cycle-dependent neuronal death. Cell-cycle-dependent neuronal apoptosis has generally been thought of as a cellautonomous process, as suggested by investigations in cell culture paradigms. However, there may be critical non cell-autonomous contributors to this process in vivo. The CNS phenotype of mice null for the tumor suppressor pRb (pRb-1 -/-) is a striking example of such a possibility.

pRb-1-/-mice die in embryonic life (E13-E15) with significant hematopoietic abnormalities and massive apoptotic neuronal death in the central and peripheral nervous system [15,16]. The Interestingly, more recent studies have indicated that central neuronal apoptosis in pRb-1-/-mice is not cell-autonomous. In chimeric mice, composed of wild-type and pRb-1-/-cells, central (but not peripheral) neuronal apoptosis does not occur despite the failure to achieve cell-cycle exit, indicated by abnormal tetraploid cells. The cell-cycle profile, however, of these neurons appear to be different because, while PH3-staining and FACS analyses indicate that many CNS neurons have entered late G2 or M phase in the germline pRb-1-/-animals, neurons in the chimeric mice do not appear to enter M-phase [17]. Surprisingly, the largely normal development of the CNS in chimeras implies that tetraploid neurons are perhaps even capable of differentiation. Thus, these results indicate that apoptosis in this particular developmental context is non cell-autonomous and that failure to exit the cell cycle is not sufficient to trigger central neuronal apoptosis. The results also raise the possibility that apoptosis is triggered at the G2/M checkpoint and that non cell-autonomous factors are required to drive neurons into late G2/M. Finally, the studies indicate important differences between cell-death signaling in central and peripheral nervous system neurons. An intriguing recent study has indicated that the critical non cell-autonomous factors that trigger apoptosis in this model may be placentally derived since pRb-1-/-mice grown in a wild-type placenta do not develop CNS pathology [68,69].

Drosophila is an attractive model system in which to search for potential non cellautonomous factors in cell-cycle-dependent neuronal death. For example, in addition to ectopic neuronal expression of cyclins or E2f/Dp, it would be interesting to introduce other genetic or environmental manipulations, both within and outside the nervous system, to determine factors that might modulate the neuronal response to ectopic cell-cycle activation. Obvious candidates for such manipulations would be oxidative stress, DNA damage and cell-cycle checkpoint

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pathways, all of which have been extensively characterized in a variety of contexts in flies [70,71]. The modulation of molecules known to alter lifespan, such as those in the insulin signaling pathway or Sir2, within the brain and fat body might also be very interesting in this context [72][73][74]. In addition, genetic tools are now available in flies to facilitate creation and analysis of clones in the brain, allowing ectopic activation of the cell cycle in a select group of neurons, or in glial cells. It would be interesting if neuronal cell-cycle activation can lead to non cellautonomous death of surrounding neurons, potentially analogous to the non cell-autonomous apoptosis that occurs in cells surrounding clones that have been conferred a growth advantage.

Several recent papers have been published on this phenomenon of "super-competition" in clonal analyses of developing fly imaginal discs [75,76].

How do the TOR/cell-cycle pathways become activated in the Drosophila tauopathy model?

In Section 3.2, we outlined several possibilities for tau-induced cell-cycle activation. As depicted in Figure 4, these included more general mechanisms, including generation of oxidative stress or DNA damage, inhibition of the ubiquitin-proteasome system. Other mechanisms depicted were more specifically related to tau, including direct tau-protein interactions and altered microtubule dynamics. We have made good progress in delineating signaling pathways, including TOR, that mediate cell-cycle activation in our model. An important aim of future studies will be to determine the mechanism whereby tau activates mitogenic signaling pathways. In cell-culture systems, oxidative stress activates numerous mitogenic signaling pathways [77] and fluctuations in redox potential may critically regulate cell-cycle proteins [78]. As noted above (Section 4. As mentioned in Section 3.2, tau is known to bind several molecules involved in mitogenic signaling. Homologs of these interacting proteins exist in flies and, in several cases, have been well characterized. Future studies may help determine whether genetic manipulations of these potential interactors alters tau-induced neurodegeneration and, i f so, whether tau physically interacts with these proteins in the fly eye or brain. Furthermore, the expression of tau mutants unable to bind these interactors may provide more definitive evidence of the role of such interacting proteins in mediating tau-induced cell-cycle activation. We, and other groups, have successfully made mutant tau constructs and expressed them in flies to elucidate various aspects of tau pathobiology [44,46]. While these studies would be significant if fruitful, they do rely on Finally, the ability of multiple and diverse insults to mediate neuronal cell-cycle re-entry would imply that the ability to induce cell-cycle activation is not specific to tau phosphorylation [6]. Indeed, cell-cycle mechanisms have been implicated in PD also [81][82][83]. While we did not find evidence for cell-cycle activation in tau-independent models of neurodegeneration (MJD and PD), other Drosophila models, notably recent models of AD based on ectopic beta-amyloid expression [84][85][86][87], remain to be tested for genetic modification by cell-cycle and mitogenic signaling modulation.

Conclusion

The idea, proposed more than a decade ago, that ectopic cell-cycle activation in neurons might lead not to their proliferation, but to their demise, is an intriguing one. A number of investigators subsequently identified aberrant expression of cell-cycle markers in human postmortem tissue from a variety of neurodegenerative diseases, most notably AD and other tauopathies, thus establishing this idea as one of potential clinical relevance. Further work in cell-culture systems provided mechanistic insights into how cell-cycle mediators might transduce apoptotic signals.

Recent investigations in a Drosophila tauopathy model have complemented these studies, principally by enabling a thorough genetic substantiation of the idea that cell-cycle activation mediates neuronal death in vivo. In the tauopathy model, inroads have been made in identifying signaling pathways, including TOR kinase signaling, that may mediate aberrant mitogenic signals in the human disease. These investigations raise the possibility that TOR and the cell cycle are therapeutic targets in human tauopathies, including AD, a hypothesis that is now amenable to testing in mouse tauopathy models. Powerful genetic methods available in flies,

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including the ability to express mutant tau constructs, have enabled mechanistic insights to be made. For example, the experiments investigating the epistasis of tau phosphorylation to cellcycle activation suggest that tau phosphorylation is upstream of cell-cycle activation [46].

For all the progress that has been made in human tissue, mammalian cell-culture and Drosophila model systems, fundamental and important questions remain. These include whether

or not a coordinated cell-cycle activation, or simply certain cell-cycle activators, are required to mediate neuronal death; whether ectopic neuronal cell-cycle activation is always sufficient to mediate neuronal apoptosis; and whether cell-autonomous versus non cell-autonomous factors play a role in cell-cycle-mediated neurodegeneration. Finally, the relationship of cell-cycle activation to other signaling pathways, including those related to cell-cycle checkpoints and aging, remain unclear in vivo.

It is intriguing that there may be a significant mechanistic connection between oncogenesis and neurodegeneration, two major pathologies associated with aging. With versatile and powerful tools available, Drosophila models may be superbly suited to gauge the depth of this connection, and its basis. (photoreceptors and support cells). F and G have been reproduced with permission from [42]. [67,88,89], oxidative stress [66] and dysfunction of the ubiquitin-proteasome system [80]. Other mechanisms may be tau-specific, including direct-tau protein interactions that subserve activation of mitogenic signaling pathways, and indirect activation of the cell cycle through alterations of microtubules.

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  remained an important open question amenable to genetic analysis. In the fly tauopathy model, reducing tau phosphorylation genetically, by reducing the level of a known tau kinase glycogen A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10 synthase kinase-3 (GSK-3), led to decreased cell-cycle activation. Conversely, a pseudophosphorylated tau construct exhibited dramatically increased toxicity and commensurately increased ectopic cell-cycle marker expression in the brain. Furthermore, the toxicity induced by this pseudophorylated construct was blocked by cell-cycle inhibition. Finally, immunohistochemical data indicated that only a small subset of neurons containing abnormally phosphorylated tau were immunopositive for cell-cycle markers [46]. Taken together, these experiments indicated that cell-cycle activation occurred downstream of abnormal tau phosphorylation in the fly model. In our view, these experiments exemplify the utility of simple genetic models in casting light upon mechanistic questions that remain difficult to resolve in other model systems, or in human postmortem tissue analysis. Of course, the findings in the fly model do not rule out alternative mechanisms operating in mammalian models or in human disease.

  transducing complex signals through binding multiple regulatory and adapter proteins. The receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor, are classic example of such molecules. RTKs have tyrosine kinase catalytic function but contain multiple tyrosines that, when phosphorylated, bind SH2 domain-containing proteins including the adapter protein PLC-γ and Src family tyrosine kinases. Such interactions serve to amplify mitogenic signals by providing additional docking sites for modular signaling proteins.

14 apoptosis 4 . 1 "

 1441 First and foremost, a causal connection has been established between cell-cycle mediator activation and tau-induced neurodegeneration in vivo. Second, in the fly model, genetic manipulations and the use of different tau constructs has been useful in determining the epistasis of tau phosphorylation to cell-cycle activation. Our data would indicate that tau phosphorylation is upstream of cellcycle activation, Third, we have shown that TOR signaling is required for tau-induced neurodegeneration and that TOR can ectopically activate cell cycle-dependent neuronal A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT in vivo. Fundamental questions remain more open, however, and these will be discussed in turn. Cell-cycle activation" in neurodegeneration: do we mean a coordinated cell cycle or cell-cycle mediator-dependent apoptosis? Our genetic analyses in the tauopathy model indicate that activation of cell-cycle mediators is necessary for tau-induced neurodegeneration, since numerous manipulations at both the G1/S and G2/M checkpoints with multiple genetic reagents produce a consistent pattern of modification [46]. The presence of both PCNA and PH3, markers of different cell-cycle phases, supports the possibility that neurons may have re-entered a coordinated cell cycle. Certainly, the absence of any evidence of cytokinesis in our model indicates that neurons cannot successfully progress through mitosis. Our genetics would also generally indicate stronger effects of modulating G2/M cyclins on tau phenotypes than G1/S cyclins (see Section 3.1, above), indirectly supporting neuronal apoptosis being transduced later in the cell cycle. We cannot, however, rule out a second possibility that positive cell-cycle regulators, including Cdks and E2f1/Dp, are involved in transducing tau-induced apoptosis in the absence of a coordinated cellcycle progression. Studies in cell-culture systems have revealed that E2F family transcription

  only available read-out for cell-cycle activation in the fly brain. Each marker could, for example, represent multiple phases of the cell cycle (PCNA: G1, S, G2; PH3: G2, M), a transcriptional upregulation of cell-cycle regulators in the absence of co-ordinated cell-cycle activation, an immediate-early gene response (PH3) or a response to DNA damage (PCNA). Again, accurate determination of cell-cycle phasing in different contexts may help resolve these issues because it may be that neuronal apoptosis is transduced at the G2/M and not the G1/S transition in vivo. It is possible that this is context-dependent and dependent upon pathogenesis, or even variable among different populations of neurons exposed to the stimulus. Indeed, in flies, as described in Section 3.1, ectopic expression of different cyclins leads to cell-cycle marker expression and apoptosis in different neuronal populations.

  appearance of the neuronal phenotype temporally coincides with the normal neuronal cell-cycle exit and commencement of differentiation. Histologically, proliferation-competent neurons in the ventricular zone of the neural tube are normal but apoptotic and mitotic markers are found in CNS regions usually populated by postmtitotic cells. These findings imply a role for pRb-1 in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 18 either establishing or maintaining the postmitotic state, and possibly in neuronal differentiation.

  2), we have recently found that genetically and pharmacologically modulating the oxidative stress A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 20 response can modify tau-induced neurodegeneration phenotypes (Dias et al., in press). In fact, decreasing anti-oxidant defences in the fly brain does potentiate tau's ability to induce cell-cycle marker re-expression, implicating oxidative stress in this process. Interestingly, microtubule stabilization in proliferating can induce TOR-dependent apoptosis [79]. It will be interesting to determine whether direct manipulations of the microtubules, whether genetic or pharmacologic, can recapitulate cell-cycle phenotypes, Likewise, numerous genetic reagents are now available in Drosophila to manipulate the UPS and thus the hypothesis that defective degradation of mitotic cyclins leads to aberrant cell-cycle activation [80] is readily testable in vivo in flies.

  limited candidate-based testing based on in vitro tau-protein interactions, or on predictions of potential tau interactors based on modular domains that may not hold true in vivo. Unbiased genetic screens and protein interaction studies, such as yeast-2-hybrid or immunoprecipitation in conjunction with mass spectrometry, may represent powerful alternative approaches. In addition, libraries for many modular domains now exist. It may be worthwhile to test tau for binding in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 21 these libraries and to verify interesting candidates for their ability to mediate tau-induced cellcycle activation in vivo.

Figure 1 :

 1 Figure 1: Schematic diagram contrasting different consequences of cell-cycle activation for

Figure 2 :Figure 3 :

 23 Figure 2: Simplified representation of cell-cycle regulators in mammalian (blue) and

Figure 4 :

 4 Figure 4: Potential mechanisms for tau-induced cell-cycle activation. These include general

Figure 5 :

 5 Figure 5: Drawing analogies between tau and receptor tyrosine kinases. Tau contains 14

Acknowledgments

The authors would like to thank Ilan Elson-Schwab for useful discussions and his thoughtful comments on the manuscript.

ACCEPTED MANUSCRIPT

29 bind to SH2/3-containing proteins including Src family non-receptor tyrosine kinases [51], c-Abl [90] and PLC-γ [91,92]. Binding of tau to the Fyn-SH3 domain occurs at the PXPXXP sequence shown (232-236). Tau also binds the WW domain-containing protein Pin-1, which isomerizes P-Thr-231 to render it more readily dephosphorylated by PP2A [93]. Other candidate proteins of interest, with genetic reagents available in flies, include the WW domain-containing proteins hWW-45 and Itch and the SH2/SH3-containing RASGAP. Note that fly homologs are labeled in black.

Figure 6: TOR signaling in Drosophila [94][95][96]. TOR integrates nutrient availability with signals from multiple pathways, including insulin signaling/PI3K. The insulin receptor is a ~400kDa tyrosine kinase receptor. PI3K is activated by direct recruitment to the insulin receptor substrate (IRS; also known as Chico). In mammalian systems, a well-characterized mechanism of PI3K activation is through Ras. PI3K appears to activate MAP kinase pathways in mammalian cells, but this does not appear to be the case in flies [96]. However, signaling from PI3K to Akt, and to TOR is well conserved. Akt phosphorylates and inhibits Tsc2 [97], the GAP protein inhibitory for the Rheb GTPase [98]. Rheb activates TOR and drives growth and indirectly the G1/S transition [99][100][101]. Over-expressing TOR itself paradoxically downregulates TOR signaling [102]. The two major downstream effectors are S6k and eIF4e. In flies, TOR activates Cyclin D and the Myc oncogene [61]. eIF4e is in turn a transcriptional target of Myc [104]. Information regarding specific genetic reagents is published [46] or is available from the authors by request.

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Christina J Sigurdson

Adriano Aguzzi

Chronic wasting disease

Until recently, chronic wasting disease of cervids, the only wildlife prion disease, was believed to be geographically concentrated to Colorado and Wyoming within the United States. However, increased surveillance has unveiled several additional pockets of CWD-infected deer and elk in 12 additional states and 2 Canadian provinces. Deer and elk with CWD have extensive aggregates of PrP Sc not only in the central nervous system, but also in peripheral lymphoid tissues, skeletal muscle, and other organs, perhaps influencing prion shedding. Indeed, CWD is transmitted efficiently among animals by horizontal routes, although the mechanism of spread is unknown. Genetic polymorphisms in the Prnp gene may affect CWD susceptibility, particularly at codon 225 (S/F) in deer and codon 132 (M/L) in elk. Since CWD infects free-ranging animals and is efficiently spread, disease management will be a challenge.

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3

A chronicle of CWD:

A prion disease of free-ranging wildlife, chronic wasting disease (CWD) affects mule deer (Odocoileus hemionus), white-tailed deer (O. virginianus), Rocky Mountain elk (Cervus elaphus nelsoni) [START_REF] Williams | Chronic wasting disease in deer and elk in North America[END_REF], and moose (Alces alces shirasi) [START_REF] Williams | Chronic wasting disease[END_REF], all members of the family Cervidae. CWD was first noted in 1967 within a research facility in Fort Collins, Colorado where captive mule deer used for nutrition research were reported with a body wasting syndrome [START_REF] Williams | Chronic wasting disease[END_REF]. After more than a decade of uncertainty about the etiology of CWD, pathologists Elizabeth Williams and Stewart Young recognized the brain lesions as those of a transmissible spongiform encephalopathy (TSE) in 1978, and CWD was subsequently demonstrated as a prion disease not only by the classic neuronal perikaryonic vacuoles [START_REF] Williams | Chronic wasting disease of captive mule deer: a spongiform encephalopathy[END_REF], but also by the accumulation of aggregated prion protein [START_REF] Spraker | Comparison of histological lesions and immunohistochemical staining of proteinase-resistant prion protein in a naturally occurring spongiform encephalopathy of free-ranging mule deer (Odocoileus hemionus) with those of chronic wasting disease of captive mule deer[END_REF] (Fig. 1) as well as prion infectivity in the brain [START_REF] Browning | Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP[END_REF]. In the late 70s and early 80s, CWD was detected in two zoological collections, in Wyoming and in Canada [START_REF] Williams | Spongiform encephalopathies in Cervidae[END_REF].

Beginning in 1981, cases of CWD were discovered in wild deer and elk on the eastern slope of the Rocky Mountains and extending out on the plains following river valleys within Colorado and Wyoming [START_REF] Spraker | Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado[END_REF][START_REF] Miller | Epizootiology of chronic wasting disease in freeranging cervids in Colorado and Wyoming[END_REF]. By 1996, CWD was first detected in Canada's farmed elk, and soon thereafter in the US elk industry, although it may have occurred in this industry far earlier. More recently, CWD-infected ranched elk have been discovered in several other US states and in South Korea [START_REF] Watts | The expanding universe of prion diseases[END_REF] [10] raising international awareness and concern regarding CWD. The origin of CWD remains an enigma.

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Epidemiology and disease management:

Based on published and unpublished estimates, there may be well over 30 million cervids in North America [START_REF] Kong | Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models[END_REF]. Prior to 2000, it was known that CWD had spread in part through transport of captive deer and elk and movements of free-ranging animals, but its distribution was believed to be limited to a 40,000km 2 region of northern Colorado and southern Wyoming [START_REF] Williams | Chronic wasting disease in deer and elk in North America[END_REF][START_REF] Miller | Epizootiology of chronic wasting disease in freeranging cervids in Colorado and Wyoming[END_REF], with a small number of cases in Canada [START_REF] Williams | Chronic wasting disease in deer and elk in North America[END_REF][START_REF] Williams | Spongiform encephalopathies in Cervidae[END_REF]. CWD surveillance has recently been undertaken in other states and provinces, and the results have been astonishing. CWD-infected cervids have been reported in 12 additional states, extending east to New York and West Virginia, as well as in 2 Canadian provinces ( http://www.aphis.usda.gov/vs/nahps/cwd/cwddistribution.html;http://www.nwhc.usgs.gov/disease_information/chronic_wasting_dis ease/north_america_CWD_map.jsp) (Fig. 2). The distribution pattern is not in contiguous zones consistent with natural movement of free-ranging animals, but instead concentrated in focal hotspots of varied size separated by large distances (Fig. 2). Wisconsin has dense white-tailed deer populations (15-20 deer/km2) with a prevalence of up to 13% of the male deer in some regions [START_REF] Grear | Demographic patterns and harvest vulnerability of chronic wasting disease infected white-tailed deer in Wisconsin[END_REF]. The origins of these recent outbreaks remain under investigation, b ut in some cases spillover from infected game farms seems a plausible explanation. The appearance of CWD in wild cervids presents significant challenges to disease control or eradication due to (i) the extensive geographic range of North American deer and elk, (ii) the logistical difficulty in applying ante-mortem diagnostic tests such as tonsil biopsy [START_REF] Wolfe | Evaluation of antemortem sampling to estimate chronic wasting disease prevalence in free-ranging mule deer[END_REF], and (iii) the inability to rid the environment of potential prion contaminated excreta.

CWD surveillance in Europe has been more limited, however some countries such as Germany have conducted an active surveillance program. In Germany, a total of

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7300 captive and free-ranging cervids were tested for CWD with no sign of infection [START_REF] Schettler | Surveillance for prion disease in cervids, Germany[END_REF]. Reindeer or caribou (Rangifer tarandus), from North America or Northern

Europe respectively, have a highly homologous prion sequence compared with mule deer, thus are likely susceptible to CWD. Other European cervids such as moose and red deer (Cervus elaphus) are also expected to be CWD-susceptible.

CWD infects free-ranging animals, creating an enormously complex situation for controlling disease spread, particularly in light of our poor understanding of specific transmission routes and susceptibility of non-cervid species. In addition, prioninfected deer and elk will be consumed by scavengers and other carnivores, including mountain lions, foxes, raccoons, coyotes, as well as eagles and vultures.

Domestic ruminants and other herbivores are likely exposed through CWD contaminated grazing areas, and conversely, wild ruminants are likely exposed to sheep scrapie. Species known to be susceptible to CWD by an extreme and unnatural exposure route, intracerebral inoculation, include ferrets [START_REF] Bartz | The host range of chronic wasting disease is altered on passage in ferrets[END_REF], raccoons [START_REF] Hamir | Experimental inoculation of scrapie and chronic wasting disease agents in raccoons (Procyon lotor)[END_REF], other ruminants (discussed below), and squirrel monkeys [START_REF] Marsh | Interspecies transmission of chronic wasting disease prions to squirrel monkeys (Saimiri sciureus)[END_REF]. Studies are ongoing to determine whether mountain lions (Puma concolor) are susceptible (M.

Miller, pers. communic. and [START_REF] Wolfe | Suspected secondary thiafentanil intoxication in a captive mountain lion (Puma concolor)[END_REF]).

Transmission among cervids:

Of all the mammalian prion diseases, CWD is likely the most efficiently transmitted. In dense free-ranging deer populations, CWD prevalence can reach as high as 30%, however in captive herds, prevalence can climb to nearly 100% [START_REF] Williams | Chronic wasting disease[END_REF]. How is CWD transmitted with such efficiency? This question is arguably one of the biggest conundrums in the CWD field, and hypotheses range from spread via direct contact

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to exposure through grazing in areas contaminated by prion-infected secretions, excretions (saliva, urine, feces), tissues (placenta), or decomposed carcasses.

Indeed, Miller et al. have shown that CWD-infected carcasses allowed to decay naturally in confined pastures can lead to CWD infections in captive deer [START_REF] Miller | Environmental sources of prion transmission in mule deer[END_REF].

Perhaps multiple exposure pathways can lead to an infection, nevertheless, horizontal spread of CWD is clearly occurring [START_REF] Miller | Prion disease: horizontal prion transmission in mule deer[END_REF] and vertical transmission cannot be excluded. Conceivably the abundant PrP Sc in tonsils and Peyer's patches [START_REF] Spraker | Comparison of histological lesions and immunohistochemical staining of proteinase-resistant prion protein in a naturally occurring spongiform encephalopathy of free-ranging mule deer (Odocoileus hemionus) with those of chronic wasting disease of captive mule deer[END_REF] contributes to PrP Sc shedding in saliva or feces. Nonetheless, environmental prion contamination underscores the difficulties of CWD eradication, a goal which is unlikely to be met for free-ranging cervid populations with the available management techniques. Indeed, CWD eradication is not a stated management goal for all western states.

Inflammation may increase the risk of prion shedding in cervids. In prion-infected mice, follicular inflammation in the kidney directs prions to accumulate within lymphoid follicles [START_REF] Heikenwalder | Chronic lymphocytic inflammation specifies the organ tropism of prions[END_REF] and intriguingly, leads to prion excretion into the urine of infected mice [START_REF] Seeger | Coincident scrapie infection and nephritis lead to urinary prion excretion[END_REF]. Even in natural sheep scrapie cases, follicular mastitis results in prion accumulation in the mammary gland [START_REF] Ligios | PrPSc in mammary glands of sheep affected by scrapie and mastitis[END_REF]. It remains to be seen whether scrapie prions are then shed into the milk to infect nursing lambs. Because deer and elk also have a widespread prion assemblage within lymphoid tissues, it seems plausible that follicular inflammation may also lead to CWD prion build-up in nonlymphoid organs, potentially shifting shedding routes. It is unknown whether other types of inflammation, such as the granulomatous inflammation in the intestine seen in Johne's disease (Mycobacterium avium subsp. paratuberculosis) (affects ruminants, including deer and elk) or parasitic inflammation could lead to or perhaps increase prion excretion by fecal routes.

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7

Recent studies of prion disease in hamsters indicate the potential for prion shedding via saliva [START_REF] Dejoia | Prion infection of oral and nasal mucosa[END_REF]. In hamsters intracerebrally (ic) exposed to scrapie, prions are transported centrifugally from brain to the tongue, and PrP Sc deposits in muscle, nerve, taste buds, and epithelium, serving as a large potential reservoir for continual PrP Sc shedding into saliva. In addition, PrP Sc has been detected in the tongues of 7 of 10 sheep naturally infected with scrapie using b oth western blotting and immunohistochemistry techniques [START_REF] Casalone | Pathological prion protein in the tongues of sheep infected with naturally occurring scrapie[END_REF]. However, the tongue of CWD infected deer and elk has not yet been investigated for the presence of PrP Sc or prion infectivity.

Nevertheless, CWD-infected tonsils contain abundant PrP Sc (Fig. 1) [START_REF] O'rourke | Abundant PrP(CWD) in tonsil from mule deer with preclinical chronic wasting disease[END_REF] and may also serve as a source for prion shedding into saliva. In light of the commonly shared salt licks and water sources in captivity, as well as licking behaviors of deer and elk, PrP Sc transmission via saliva should be considered as suspect.

Transmission to livestock:

The capacity for CWD transmission to other species is clearly an area of great concern since potentially CWD-infected free-ranging animals are co-habitating with domestic ruminants. However, data on the risk for other wildlife species or domestic ruminants contracting the disease is steadily accumulating. Cattle have been challenged with CWD by 3 routes: (i) intracerebral, (ii) oral, and (iii) via contact exposure to CWD-infected mule deer (co-habitation)[25, M. Miller, pers. communic].

After 6 years, only cattle challenged by ic inoculation have developed disease. Five of thirteen animals (38%) developed prion infection after an incubation period of 2-5 years [START_REF] Hamir | Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route[END_REF]. Secondary passage of the cattle CWD led to a decrease in incubation period to ~16 months with 100% attack rate (n=6) [START_REF] Hamir | Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle[END_REF]. Perhaps surprisingly, cattle [START_REF] Kong | Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models[END_REF][START_REF] Belay | Chronic wasting disease and potential transmission to humans[END_REF] Other indirect studies of human susceptibility to CWD, although limited in number, suggest that the risk is low. In biochemical conversion studies, Caughey et al.

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showed that the efficiency of CWD to convert recombinant human PrP into amyloid fibrils was low, but similar to that of both BSE and scrapie fibrils to do the same [START_REF] Raymond | Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease[END_REF].

Recently Xie et al. have compared histopathology and PrP Sc b iochemical characteristics from deer and elk with that of humans with sporadic CJD cases that are methionine homozygous at codon 129 [START_REF] Xie | Chronic wasting disease of elk and deer and Creutzfeldt-Jakob disease: comparative analysis of the scrapie prion protein[END_REF]. The PrP Sc form is cleaved by proteinase-K at different sites depending on the conformation of the protein so can be used to aid determination of whether the PrP Sc conformation is similar. For CWD, the unglycosylated PK-resistant PrP Sc migrated at 21 kDa, similar to sCJD (MM1 subtype), the PK cleavage site was the same, occurring at residues 78 and 82 as assessed by N-terminal sequencing, and the conformational stability also showed no significant difference between elk CWD and sCJD MM1 cases. However, there were distinct glycoform patterns exhibited by two dimensional gel electrophoresis, suggesting that the elk CWD and human sCJD MM1 strains differ, although strain features, including histologic profile, target organs, and glycoform patterns, will not necessarily remain the same upon crossing species barriers [START_REF] Bartz | The host range of chronic wasting disease is altered on passage in ferrets[END_REF][START_REF] Bellworthy | Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge[END_REF][START_REF] Nonno | Efficient transmission and characterization of creutzfeldt-jakob disease strains in bank voles[END_REF]. or >756 days [START_REF] Kong | Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models[END_REF].

Cervid prion genetics:

The deer and elk primary protein structures are highly conserved as seen in other mammals. There are four particularly intriguing features of the deer and elk prion gene. First, a polymorphism at codon 225 (S/F) may influence CWD susceptibility.

When comparing the frequency of genotypes among CWD negative and positive free-ranging mule deer (n=1482), the odds that a CWD-infected animal was 225SS was 30 times greater when compared to 225SF [START_REF] Jewell | Low frequency of PrP genotype 225SF among free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease[END_REF].

Second, elk have a polymorphism at codon 132 (M/L) of Prnp, corresponding to polymorphic codon 129 (M/V) in humans. Elk expressing 132MM and 132ML Prnp were reported to be overrepresented among elk with CWD when compared to uninfected controls [START_REF] O'rourke | PrP genotypes of captive and freeranging Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease[END_REF], and 132LL elk experimentally infected with CWD have resisted infection for at least 4 years, whereas 132MM or 132ML elk (n=2 each) developed terminal clinical prion disease by 23 or 40 months post-inoculation, respectively, confirmed by immunohistochemistry and western blot for PrP Sc [START_REF] Hamir | Preliminary observations of genetic susceptibility of elk (Cervus elaphus nelsoni) to chronic wasting disease by experimental oral inoculation[END_REF].

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Third, white-tailed deer also have Prnp polymorphisms which may affect CWD susceptibility. When the allelic frequencies from CWD-positive and CWD-negative free-ranging, Wisconsin white-tailed deer were compared, a G96S and a Q95H polymorphism were linked to a reduced susceptibility to CWD [START_REF] Johnson | Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease[END_REF].

A fourth interesting feature of deer and elk prion genetics from an evolutionary perspective is the pseudogene which has been described in mule deer [START_REF] Brayton | A processed pseudogene contributes to apparent mule deer prion gene heterogeneity[END_REF] and white-tailed deer [START_REF] Johnson | Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease[END_REF]42]. The pseudogene is suspected to be a processed

Cervid PrP structure:

The structure of the elk prion protein has been solved by NMR analysis of recombinant elk PrP [START_REF] Gossert | Prion protein NMR structures of elk and of mouse/elk hybrids[END_REF] (Fig. 3). When the elk PrP structure is compared to human or bovine PrP structures, the global architecture is nearly identical. However, intriguingly, the elk PrP possesses an extremely well-defined loop connecting the 2 nd alpha helix and beta sheet (amino acids 166-175), whereas the homologous region is flexibly disordered in human and bovine PrP C . This loop region has been studied in detail in the laboratory of Kurt Wüthrich, as there is an outstandingly high incidence of nonconservative species variation and the loop is thought to provide structural insights into species barriers for prion disease [START_REF] Calzolai | NMR structures of three single-residue variants of the human prion protein[END_REF]. Further structural studies in 2

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mutant mouse PrP variants derived from the elk PrP primary structure, mPrP[N174T]

and mPrP[S170N, N174T], have confirmed that the defined loop in the elk is due to 2 amino acid exchanges, as the mPrP[S170N, N174T] has the conformationally identical rigid loop of the elk. Whether this loop region confers any TSE susceptibility or pathologic consequences remains to be established.

Structural differences clearly influence species susceptibility, a feature well known for sheep scrapie where susceptibility is heavily influenced by genotype at codons 136 (V/A), 154 (H/R), 171 (Q/R) [START_REF] Baylis | Study on scrapie resistance [letter[END_REF]. One proposed etiology of prion disease is based on a natural propensity for PrP to assume beta-sheet-rich conformations, in combination with a failure to prevent the accumulation of the beta-rich isoform, thereby ultimately leading to aggregated PrP [START_REF] Weissmann | The state of the prion[END_REF][START_REF] Chiti | Designing conditions for in vitro formation of amyloid protofilaments and fibrils[END_REF]. Interestingly, the amyloidogenic site of the yeast prion protein, Sup35, has the same amino acids as in the loop region of elk PrP, only the sequence order is different, but possibly suggests an inherent propensity for a beta-rich conformation with these particular amino acids [START_REF] Nelson | Structure of the cross-beta spine of amyloid-like fibrils[END_REF]. Based on this unusual rigid structural feature of the elk PrP, it is tempting to speculate that the isolated geographic foci of CWD outbreaks across the U.S. and Canada may be due to increased risk for a sporadic disease that can then spread horizontally. At least such

possibilities should remain open to discussion. Another possibility for the isolated outbreaks would be the undetected spread of CWD through commerce of captive cervids.

Clinical disease and lesions in natural cases:

Deer and elk with CWD show subtle signs of disease, characterized by weight loss, isolation from the herd, hypersalivation, polydipsia/polyuria, frequent regurgitation ±

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esophageal distension, and rarely ataxia [49] (Fig. 4). The clinical signs are nonspecific and difficult to detect in early stages except by those working daily with the animals. In one report, a captive herd of 133 white-tailed deer was discovered with 50% CWD positive in brain or lymphoid tissue, though no clinical signs were noted by the landowner or hunters [42]. The clinical course varies from several weeks to up to many months [START_REF] Williams | Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni)[END_REF].

Necropsy findings include subacute to chronic bronchopneumonia (likely due to aspiration), froth or watery rumen contents (often containing sand), abomasal or omasal ulcers, serous atrophy of bone marrow and pericardial fat, enlarged adrenal glands, and muscle atrophy [START_REF] Spraker | Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado[END_REF].

Histopathologic lesions in the brain are similar to those described for BSE in cattle and scrapie in sheep and goats: perikaryonic neuronal vacuoles, microcavitation of gray matter, astrogliosis, neuronal degeneration and loss, and PrP Sc positively labeled prion deposits and plaques. Target areas for severe spongiform change included olfactory bulb and stria, septal nuclei, thalamus, supraoptic and paraventricular nuclei, tegmental nuclei, and within the medulla oblongata, neurons of the reticular formation, as well as nuclei from the hypoglossal, vagal, medial and lateral cuneatus, and spinal tract of the trigeminal nerve [START_REF] Spraker | Comparison of histological lesions and immunohistochemical staining of proteinase-resistant prion protein in a naturally occurring spongiform encephalopathy of free-ranging mule deer (Odocoileus hemionus) with those of chronic wasting disease of captive mule deer[END_REF][START_REF] Spraker | Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado[END_REF][START_REF] Williams | Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni)[END_REF]. Neuropathology varies slightly between deer and elk: elk have more severe lesions in the thalamus and in some white matter areas [START_REF] Williams | Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni)[END_REF]. Congo red bifringent, PAS-positive amyloid plaques have been seen in deer brain but not elk [START_REF] Williams | Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni)[END_REF].

Pathogenesis:

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Transmission of CWD into deer and elk demonstrated CWD as a prion disease, with spongiform change and PrP amyloid fibrils in the brain of inoculated animals [START_REF] Williams | Chronic wasting disease[END_REF][START_REF] Williams | Spongiform encephalopathies in Cervidae[END_REF].

Oral CWD infection of mule deer fawns indicates that PrP Sc appears at early timepoints in the retropharyngeal lymph node, tonsil, and Peyer's patches by 3 months post feeding [START_REF] Sigurdson | Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus)[END_REF]. At terminal stages of disease, PrP Sc accumulates in part in the intestinal plexi and vagus nerve, although the timing of prion infection of ganglia and nerves is not known [START_REF] Sigurdson | PrP(CWD) in the myenteric plexus, vagosympathetic trunk and endocrine glands of deer with chronic wasting disease[END_REF].

CWD is one of several prion diseases in which PrP Sc is typically disseminated throughout the lymphoid system prior to the CNS. Thus, tonsil biopsy can be used to diagnose CWD ante-mortem, although a negative tonsil biopsy does not rule out CWD infection as not all follicles are necessarily positive [START_REF] Sigurdson | Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus)[END_REF][START_REF] Wild | Preclinical diagnosis of chronic wasting disease in captive mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) using tonsillar biopsy[END_REF] and not all CWD cases have a lymphoid phase of infection. This scenario is seen particularly in elk, where 28/226 CWD positive elk in one study did not have detectable PrP Sc in the lymphoid system [START_REF] Spraker | Variable patterns of distribution of PrP(CWD) in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease[END_REF]. PrP Sc in lymphoid tissue co-localizes with follicular dendritic cell (FDC) and tingible body macrophage markers [START_REF] Sigurdson | PrP(CWD) lymphoid cell targets in early and advanced chronic wasting disease of mule deer[END_REF], as seen with natural sheep scrapie and experimental mouse scrapie [START_REF] Heggebo | Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep[END_REF][START_REF] Mcbride | PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice[END_REF]. In mouse scrapie models, C1q is essential for peripheral PrP Sc replication, and C1q deficiency leads to a marked delay in incubation period, suggesting that the classical complement cascade is an important early step in prion pathogenesis [START_REF] Klein | Complement facilitates early prion pathogenesis[END_REF][START_REF] Mabbott | Temporary depletion of complement component C3 or genetic deficiency of C1q significantly delays onset of scrapie[END_REF]. The mechanism by which C1q interacts with PrP Sc and the connection with accumulation on FDCs is unresolved, but may also have relevance for CWD pathogenesis.

Early stages of oral uptake of prions across the intestinal mucosa of sheep have been recently examined in greater detail by Jeffrey et al [START_REF] Jeffrey | Transportation of prion protein across the intestinal mucosa of scrapiesusceptible and scrapie-resistant sheep[END_REF]. Loops of intestine were isolated and inoculated with prions. PrP Sc was detected in the dilated villous lacteals PrP Sc in mule deer include adrenal glands, pancreatic islets, and pituitary [START_REF] Sigurdson | PrP(CWD) in the myenteric plexus, vagosympathetic trunk and endocrine glands of deer with chronic wasting disease[END_REF].

New tools for CWD investigation:

A big leap forward for CWD research occurred with the development of cervid PrP expressing transgenic mice (CerTgPrP), recently been described by Browning et al.

to be highly susceptible to CWD infection [START_REF] Browning | Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP[END_REF]. Mice develop plaques in the brain resembling CWD in cervids. Thus far, prion infectivity in various organs of cervids has not yet been examined in detail by bioassay, however, CWD infectivity has been

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recently shown in skeletal muscle of naturally CWD-infected deer [START_REF] Angers | Prions in Skeletal Muscles of Deer with Chronic Wasting Disease[END_REF]. A second cervid PrP transgenic mouse has been described by Kong et al. [START_REF] Kong | Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models[END_REF].

A CWD-susceptible cell line has been developed derived from cervid brain fibroblasts, and has been used to screen inhibitors of CWD infection, e.g., pentosan polysulfate [START_REF] Raymond | Inhibition of protease-resistant prion protein formation in a transformed deer cell line infected with chronic wasting disease[END_REF]. This is the first CWD specific assay introduced for screening compounds that inhibit CWD propagation. Perhaps these cells will also be useful to assess CWD infectivity.

CWD strains?

In sheep scrapie, many strains of TSEs exist, including an 'atypical scrapie' known as Nor98 [START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type, Nor98[END_REF]. For CWD, the existence of multiple strains is unclear. Transgenic mice overexpressing murine PrP and inoculated with CWD develop a mixture of pathological and biochemical phenotypes which may suggest the existence of multiple strains, although the phenotypic variability could also be due to strain adaptation (CJS and AA, unpublished data). Safar et al. reported differing conformational characteristics for CWD from WTD and elk using a conformation dependent assay (CDI), also suggestive of strain differences [START_REF] Safar | Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice[END_REF].

In light of the discussion herein, one aspect of CWD research is crystal clear: there are many unsolved mysteries in this disease of wild animals. The sooner we understand basic disease factors such as CWD origins, mechanisms of spread, and species susceptibility, the more specifically we can target prevention and management programs.

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10 Kong

 10 et al. studied the question of human susceptibility to CWD by inoculating transgenic mice expressing human PrP or elk PrP with elk CWD. Whereas the elk PrP expressing mice developed disease after only 118-142 days post-inoculation, human PrP expressing mice (129M) did not develop any features of TSE after >657

Figure 1 .

 1 Figure legends:

Figure 2 .

 2 Figure 2. Map depicting the CWD cases detected in North America. (Map kindly

Figure 3 .

 3 Figure 3. Superposition of the mean NMR structures of the polypeptide segment

Figure 4 .

 4 Figure 4. Mule deer with clinical signs of CWD including hypersalivation, a lowered

  any strain typing done to look for the appearance of potentially subtle or unusual pathological or biochemical phenotypes of a new TSE strain. Fortunately, the autopsy rate for suspect cases is improving. At the National Prion Disease Pathology Surveillance Center at Case Western Reserve University (Cleveland, Ohio), CJD suspect cases are studied and classified by CJD subtype. Thus far, twenty-seven CJD patients who regularly consumed venison were reported to the Surveillance Center, however there have been no unusual or novel prion subtypes indicating the appearance of a new prion strain.

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	did not develop a spongiform encephalopathy, although PrP Sc was clearly detected in
	brain by immunohistochemistry and Western blot. By comparison, ic sheep scrapie
	infection in cattle resulted in 100% of cattle developing neurologic disease with PrP Sc
	R I P T deposits in the brain (9/9) [29]. A targeted surveillance of 262 older cattle from a CWD endemic area in Colorado did not reveal any indication of a TSE [30]. Sheep are also susceptible to CWD after intracerebral inoculation [2], but have not yet been challenged by oral routes. One goat developed TSE six years after CWD M A N U S C R I P T
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	8 9

inoculation and showed signs of intense pruritis and weight loss

[START_REF] Williams | Spongiform encephalopathies in Cervidae[END_REF]

. Elk have been challenged with sheep scrapie, and developed a spongiform encephalopathy with PrP Sc in the brain detected by immunohistochemistry and western blot. Intriguingly, the histologic lesions and PrP Sc deposits in the brain were indistinguishable from CWD in cervids

[START_REF] Hamir | Transmission of sheep scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation: final outcome of the experiment[END_REF]

. As far as we know, deer have not been directly challenged with sheep scrapie, however this experiment may be interesting and at least addresses whether sheep scrapie could be an origin for deer CWD.

CWD and human susceptibility:

Several million deer and elk hunters consume venison in the US and Canada and there is no doubt that people have been exposed to CWD. Human susceptibility to CWD is still unclear, although we can be somewhat reassured in that there have been no large scale outbreaks with hundreds of human TSE cases in Colorado and Wyoming, where CWD has existed for decades. That said, diagnosis of potential new TSE strains has been hampered in that, up until recently, autopsies were not performed on suspect human TSE cases in many states due to biosafety concerns. This indicates that clinical TSE diagnoses in humans were not confirmed, nor was

  and submucosal lymphatics by 3 hours post-inoculation, within dendritic-like cells in the draining lymph node by 24 hours, and in the Peyer's patches only by 30 days post-inoculation. Uptake was not affected by the Prnp genotype of the sheep. It is not known precisely when and how prion infectivity enters nerves, or whether PrP amplication in the Peyer's patches is a prerequisite for nerve entry, although Mabbott et al. have shown in mice that prion infection of Peyer's patches is irrelevant to CNS infection as early as 14 days post-oral challenge[START_REF] Mabbott | Follicular dendritic cell dedifferentiation by treatment with an inhibitor of the lymphotoxin pathway dramatically reduces scrapie susceptibility[END_REF].In studies by Spraker et al., PrP Sc accumulation in the brain has been characterized in great detail in MD, WTD, and elk, b oth captive and free-ranging[START_REF] Spraker | Comparison of histological lesions and immunohistochemical staining of proteinase-resistant prion protein in a naturally occurring spongiform encephalopathy of free-ranging mule deer (Odocoileus hemionus) with those of chronic wasting disease of captive mule deer[END_REF][START_REF] Spraker | Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado[END_REF][START_REF] Spraker | Variable patterns of distribution of PrP(CWD) in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease[END_REF].Experimental CWD time course studies are underway but have not yet been published to characterize the pathogenesis, therefore it remains unclear when and how PrP Sc reaches the brain and spinal cord. However, natural CWD cases in MD have been classified by PrP Sc and spongiform encephalopathy distribution throughout the brain[START_REF] Spraker | Distribution of protease-resistant prion protein and spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease[END_REF]. PrP Sc consistently accumulated in the brainstem, particularly the dorsal motor nucleus of the vagus. Other organs that accumulate

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Table 1 .

 1 Inter-ruminant prion transmission experiments.

							PrP Sc
					Time to		detected by
				# Infected/	terminal	Spongiform	IHC and/or
	Host	Inoculum	Exposure route	# Exposed	disease	encephalopathy	WB	Reference
	Cattle	Mule deer CWD+ brain	Intracerebral (ic)	5/13	2-5 years	Equivocal	Yes	27
	Cattle	Cattle CWD from ic infection above	Intracerebral	6/6	16.5 months	No	Yes	29
	Elk	Sheep scrapie	Intracerebral	3/6*	3-4 years	Yes	Yes	31
		*intercurrent death in 3 animals during first 2 years post-inoculation

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Acknowledgements

We thank Drs. Michael Miller and Madga Polymenidou for critical review of the manuscript.

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