deficient rods cope, which are larger in size than cones and need 100,000,000 opsins/cell.

The unaltered insiders discussed above are altered in other cancers; thus, whether abnormal or normal activity of a particular gene contributes to tumorigenesis is con-text dependent. There must also be genes that are expressed in one cell, where they counter transformation, and are naturally off in a neighbor, increasing its suscep-tibility. Discovering the basal unaltered features of different cell types that render them more cancer prone will expand the repertoire of potential therapies. Several

rior pole, but not preferentially at the fovea, and tumors also arise later in the periphery (B. Gallie, personal communication), which fits the posterior-anterior developmental wave rather than a cone origin. Cones in the outer retina synapse with interneurons in the inner nuclear layer. Tumors have been observed emerging here (Figure 1C), which may arise from displaced cones, or vari-ous interneurons or Mller glia in the inner nuclear layer. Retinoblastoma in mice also arises in the inner nuclear layer, exploiting the intrinsically high resistance of amacrine or horizontal neurons to apoptosis (Ajioka et al., 2007; Chen et al., 2004; MacPherson

oncogenic mutations in all tumor types, the next major step will be to expose their hidden accomplices.

Acknowledgments

The author thanks B. Gallie, P. Monnier, and J. Wrana for helpful comments.

RefeRences

Ajioka, I., Martins, R.A., Bayazitov, I.T., Donovan, S., Johnson, D.A., Frase, S., Cicero, S.A., Boyd, K., Zakharenko, S.S., and Dyer, M.A. (2007). Cell 131, 378390.

Chen, D., Livne-Bar, I., Vanderluit, J.L., Slack, R.S., 994 Cell 137, June 12, 2009 2009 Elsevier

labs are already testing MDM2/4 thera-peutics, and if thyroid hormone is required for cancer-promoting activity of THR2, perturbations in its transport or activation may also be helpful.

The cone phenotype of retinoblastoma suggests, but does not prove, that the dis-ease originates from cones. Passengers disembarking from a plane wearing winter coats imply a chilly origin, but equally could signify a frigid destination. Guessing where cancer starts from its end-stage appear-ance is equally ambiguous. If retinoblas-toma arises from cones, one would expect more tumors at the fovea, a cone-rich region located within the posterior (cen-tral) human retina. When newborn children with heterozygous loss of RB1 are tracked, nascent tumors do appear first at the poste-

Transmissible spongiform encephalopa-thies, or prion diseases, are neurode-generative conditions caused by prions, atypical infectious agents consisting of

Prion topologyAdriano Aguzzi1,* and Andrew D. Steele21Institute of Neuropathology, University Hospit2Division of Biology, California Institute of Tech*Correspondence: adriano.aguzzi@usz.ch (A.ADOI 10.1016/j.cell.2009.05.041

Inactivation of mahogunin, an E3prion disease. Chakrabarti and Heinactivate mahogunin, providing aInc.

et al., 2004). Emerging from one ground state and converting to another seems elaborate, but altering cell type is not oner-ous (induced pluripotent stem cells are a topical example), and transformation is cancers defining feature. Until the early response of different human retinal cells to RB1 loss and the routes adopted dur-ing progression are understood, we remain (cones or no cones) very much in the dark. Wherever it is first employed, the cone circuitry is clearly a crucial component in retinoblastoma.

Like other breakthroughs in the study of retinoblastoma, the latest milestone has significance well beyond the eye. Normal is not the same as harmless. As genome-wide sequencing projects continue onward toward the goal of uncovering all

PrPSc, a misfolded and aggregated form of the cellular prion protein (PrPC). PrPC is a cell-surface GPI-anchored glycopro-tein that is normally produced in abun-

and toxicity,*al Zurich, Zurich CH-8091, Switzerlandnology, Pasadena, CA 91125, USA.), steelea@caltech.edu (A.D.S.)

ubiquitin ligase, causes a spongifogde (2009) now report that prion p plausible explanation for certain Agochiya, M., and Bremner, R. (2004). Cancer Cell 5, 539551.

Chen, D., Opavsky, R., Pacal, M., Tanimoto, N., Wen-zel, P., Seeliger, M.W., Leone, G., and Bremner, R. (2007). PLoS Biol. 5, e179.

Corson, T.W., and Gallie, B.L. (2007). Genes Chro-mosomes Cancer 46, 617634.

Guigon, C.J., and Cheng, S.Y. (2009). IUBMB Life 61, 528536.

Laurie, N.A., Donovan, S.L., Shih, C.S., Zhang, J., Mills, N., Fuller, C., Teunisse, A., Lam, S., Ramos, Y., Mohan, A., et al. (2006). Nature 444, 6166.

MacPherson, D., Sage, J., Kim, T., Ho, D., McLaugh-lin, M.E., and Jacks, T. (2004). Genes Dev. 18, 16811694.

Ng, L., Hurley, J.B., Dierks, B., Srinivas, M., Salto, C., Vennstrom, B., Reh, T.A., and Forrest, D. (2001). Nat. Genet. 27, 9498.

Xu, X.L., Fang, Y., Lee, T.C., Forrest, D., Gregory-Ev-ans, C., Almeida, D., Liu, A., Jhanwar, S.C., Abram-son, D.H., and Cobrinik, D. (2009). Cell, this issue.

dance in brain, muscle, and the immune system (Aguzzi et al., 2008). Although we have a robust model of how prions repli-cate, we still do not understand how and

rm encephalopathy resembling roteins with aberrant topologies aspects of prion pathology.

why the central nervous system (CNS) is preferentially damaged. The relatively small amounts of PrPSc needed to dis-rupt CNS function suggest that prions target highly specific weak points in the system.

The key features of nearly all prion dis-eases are transmissibility and a foamy pattern (spongiosis) of brain tissue that is visible on histological sections. Spon-giosis is due primarily to intraneuronal vacuoles containing membrane frag-ments and, sometimes, degenerating organelles, but the etiology of this highly characteristic pathology is unclear. Much excitement surrounded the discovery that mutations in an E3 ubiquitin ligase called mahogunin cause similar spongi-form changes in the CNS of mahog-anoid mice (He et al., 2003). This sup-ported the notion that disorders of the proteasome degradation pathway may be involved in the toxicity of PrPSc. How-ever, recombinant prion protein does not undergo ubiquitination by mahogunin, and the potential link between mahog-unin and the pathogenic prion protein has remained untested.

In this issue, Chakrabarti and Hegde (2009) report a physical interaction between mahogunin and PrPC that has acquired improper topologies. Although the bulk of PrPC is turned over via the endolysosomal system, some is degraded by the proteasome (Ma et al., 2002 and references therein), which is the main degradation system for cyto-solic proteins (Figure 1). A minor patho-genic form of PrP called cyPrP, which lacks a secretory signal peptide and is constitutively released into the cytosol, is extremely neurotoxic to cerebellar neurons of transgenic mice (Ma et al., 2002). The neurotoxicity of cyPrP may be more widespread than initially appre-ciated as transgenic mice with inducible forebrain-restricted expression of cyPrP show behavioral and neuropathological phenotypes (Wang et al., 2009).

In previous work, Hegde and col-leagues noted that cyPrP accumulates in the cytosol due to its inefficient import into the endoplasmic reticulum (ER) attributable to a weak secretory sig-

nal sequence. Indeed, transgenic mice expressing PrP with a very inefficient secretory signal exhibit a mild neurode-generative phenotype (Rane et al., 2008), supporting the view that the cytosolic accumulation of PrPC can be neurotoxic. However, there has been much debate as to whether the cytosolic accumula-tion pathway is relevant to the toxicity of naturally occurring prion infections. To some extent, this discussion reflects the fact that it is extremely difficult to detect cyPrP and hence to design an experi-ment that would disprove its relevance to prion diseases.

In their new work, Chakrabarti and Hegde (2009) discover a functional

lead to neuronal damage and loss. Given that naked cyPrP is unstable unless the proteasome is inhibited pharmacologi-cally, the authors began by developing a GFP-tagged version of cyPrP that has greatly enhanced stability. Coexpression of tagged mahogunin led to a tight asso-ciation with aggregated cyPrP. Mahog-unin did not aggregate with a fragment of huntingtin, a protein involved in a differ-ent type of neurodegenerative disease, suggesting that mahogunin does not bind generically to all pathogenic protein

figure 1. the cellular fates of PrPIn the default pathway (black arrows), the mRNA encoding the normal cellular version of the prion protein (PrPC; blue) is translated by ribosomes docked at the ER and then is imported cotranslationally into the ER lumen. In the ER, a GPI anchor (orange oval) is attached to the C terminus of PrPC (purple), which will tether the protein to the plasma membrane. However, some fraction of PrP misfolds within the ER and is then retrotranslocated to the cytosol (cyPrP) to be degraded by the proteasome (green arrows). PrP can also end up in the cytosol via the pre-emptive quality control pathway in which ER stress leads to a lack of cotranslational import, and secretory proteins are translated and degraded in the cytosol (green arrows). During cotranslational import, a small fraction of PrP (CtmPrP) adopts a transmembrane conformation with the C terminus in the ER lumen and the N terminus facing the cytosol (gray arrows). CtmPrP may be retained within either the ER or the Golgi. Cytosolic PrP (cyPrP) and CtmPrP bind tightly to mahogunin (green), an E3 ubiquitin ligase that has been implicated in mediating spongiform pathology in the mouse brain.Cell 137, June 12, 2009 2009 Elsevier Inc. 995

interaction between cyPrP and mahog-unin, leading to an exciting new model for how mislocalized prion proteins can

aggregates. Another putative neurotoxic PrP species, CtmPrP, displays an atypi-cal transmembrane topology and is also

associated with mahogunin. Lysosomal morphology was altered in the same way by loss of mahogunin and by cyPrP, sug-gesting a connection between these two proteins. As no substrates of mahog-unin are known, the authors relied on lysosomal morphology as a surrogate of mahogunin function. Finally, the authors observed loss of mahogunin immunore-activity and altered lysosomal morphol-ogy in the brains of mice expressing a mutant form of PrP that favors produc-tion of CtmPrP.

The finding of bilaminar spongiosis (a pattern of vacuolation that affects two discontinuous cortical layers) in human brain specimens rings alarm bells with any neuropathologist, as it is a harbinger of the prototypic prion disease of humans, Creutzfeldt-Jakob disease. The new study is tantalizing as it suggests that in order to decipher the molecular basis of prion toxicity we may need to enumerate and functionally assess the substrates of mahogunin. However, it will be crucial to assess whether mahogunin plays a role in mediating neurodegeneration in natu-rally occurring prion diseases such as Creutzfeldt-Jakob disease and in experi-mental models of prion infection such as transmission to mice of Rocky Mountain Laboratory (RML) scrapie prions.

Although it was the spongiosis in mahoganoid mice that flagged the prion-mahogunin connection, the spongiosis phenotype raises some tough questions. Given that mahoganoid mice develop spongiosis and that both cyPrP and CtmPrP appear to sequester mahogunin, it would be gratifying to observe spon-giosis in mice overproducing cyPrP and CtmPrP. However, although neither mouse strain is healthy, spongiform changes in brain tissue are not prominent (Ma et al., 2002; Rane et al., 2008; Stewart and Harris, 2005). Conversely, spongio-sis is a hallmark of prion infections, yet the extent to which typical prion infec-tions lead to the generation of cyPrP or CtmPrP is unclear, and the effect of prion infections on the cellular bioavailability of mahogunin has not been investigated. So might spongiosis be a red-herring phenotypic similarity in mahogunin-defi-cient and prion-infected mice? Investi-996 Cell 137, June 12, 2009 2009 Elsevier

gating this issue will be important given that PrP oligomers can interfere directly with the proteolytic activity of the pro-teasome (Kristiansen et al., 2007), sug-gesting that there are mahogunin-inde-pendent pathways of PrPSc toxicity. Also, cyPrP interacts with the hydrophobic core of membranes implying that it could subvert the function of synaptic vesicles (Wang et al., 2006).

Another poorly understood aspect of prion toxicity is the role of PrPC in cellular damage. We know that PrPC is generally required for PrPSc to become neurotoxic (Brandner et al., 1996). Furthermore, in a transgenic mouse expressing a neu-rotoxic PrP mutant (3AV) with enhanced transmembrane topology, coexpression of wild-type PrPC contributes to disease progression (Stewart and Harris, 2005). However, the neurotoxicity of several other mutant forms of PrP is antago-nized effectively by wild-type PrPC. Cur-rently, there is no convincing theoretical framework (including the one sketched by Chakrabarti and Hegde) that can rec-oncile these observations.

So how might the results of Chakrabarti and Hegde instruct our understanding of neurotoxicity in prion infections? As the authors note, there is an increase in ER stress markers during prion infection, suggesting a vicious circle: misfolded PrP generated during prion infection may escape from the ER and enter the cytosol via a pre-emptive quality control pathway described in previous work by Hegde and colleagues. This may lead to mahogunin inactivation and other untow-ard events including lipid membrane dis-ruption and proteasome inhibition, which may favor the accumulation of cyPrP and prion pathogenesis.

Two key experiments are needed to directly test the relevance of the mahog-unin-cyPrP connection to prion diseases. First, if cyPrP is a significant contributor to pathogenesis in the infectious forms of prion disease, one might expect prion infection to be synergistic with cyPrP. Hence, mice expressing cyPrP should experience aggravated pathogenesis after prion infection. However, transgenic cyPrP expression shortened the latency of disease by only ~15 days in mice infected with the RML prion strain (which is marginal at best) and not at all with a different strain, 22L (W. Jackson, A.D.S., Inc.

and S. Lindquist, unpublished data). Sec-ond, if prion toxicity is mediated primarily by mahogunin, one might expect mahog-anoid mice (which are not healthy yet live long enough for the purpose of this exper-iment) to become paradoxically resistant to prion infection. However, the presence of the mahoganoid mutation did not mod-ulate the progress of RML prion infection (G. Carlson and G. Barsh, personal com-munication). These unpublished results suggest that the mahogunin-cyPrP con-nection may be less crucial for bone fide prion infection, or that mahogunin deple-tion might be compensated for by other mechanisms. Conversely, certain familial forms of human prion disease are asso-ciated with only small amounts of PrPSc but with increased amounts of CtmPrP and are more likely to fit the mechanism described by Chakrabarti and Hegde. In the long run, small molecules that target the mahogunin-cyPrP interaction may provide plausible therapeutic leads for treating the unfortunate families afflicted with these hereditary prion diseases.

Acknowledgments

A.A. is supported by the Swiss National Founda-tion, European Union FP7, and the Stammbach Foundation. A.D.S. is supported by the Broad Fel-lows in Brain Circuitry program at Caltech.

RefeRences

Aguzzi, A., Sigurdson, C., and Heikenwaelder, M. (2008). Annu. Rev. Pathol. 3, 1140.

Brandner, S., Isenmann, S., Raeber, A., Fischer, M., Sailer, A., Kobayashi, Y., Marino, S., Weissmann, C., and Aguzzi, A. (1996). Nature 379, 339343.

Chakrabarti, S., and Hegde, R.S. (2009). Cell, this issue.

He, L., Lu, X.Y., Jolly, A.F., Eldridge, A.G., Watson, S.J., Jackson, P.K., Barsh, G.S., and Gunn, T.M. (2003). Science 299, 710712.

Kristiansen, M., Deriziotis, P., Dimcheff, D.E., Jack-son, G.S., Ovaa, H., Naumann, H., Clarke, A.R., van Leeuwen, F.W., Menendez-Benito, V., Dantu-ma, N.P., et al. (2007). Mol. Cell 26, 175188.

Ma, J., Wollmann, R., and Lindquist, S. (2002). Sci-ence 298, 17811785.

Rane, N.S., Kang, S.W., Chakrabarti, O., Feigen-baum, L., and Hegde, R.S. (2008). Dev. Cell 15, 359370.

Stewart, R.S., and Harris, D.A. (2005). J. Biol. Chem. 280, 1585515864.

Wang, X., Wang, F., Arterburn, L., Wollmann, R., and Ma, J. (2006). J. Biol. Chem. 281, 1355913565.Wang, X., Bowers, S.L., Wang, F., Pu, X.A., Nel-son, R.J., and Ma, J. (2009). Biochim. Biophys. Acta. Published online March 10, 2009. 10.1016/j.bbadis.2009.02.014.

Prion Topology and ToxicityAcknowledgmentsReferences