A Potential Prion Therapy Focuses Attention on Protein Conversion

British researchers report in today’s Science that they have managed to prevent and even reverse clinical manifestations of prion disease. Surprisingly, they did it not by eliminating deposits of abnormal prions, but by depleting normal prion protein. Beyond the therapeutic implications, this result suggests that neurotoxicity in the disease is a function of the protein’s conversion process rather than its final product.

Prion diseases ranging from Creutzfeldt-Jacob to mad-cow are caused by prion proteins (PrPc) being misfolded, or so the story goes. But do these abnormal proteins (PrPSc) cause neurodegeneration? There are some bits of evidence to the contrary, even aside from the fact that some human prion diseases feature little or no detectable PrPSc, the authors write. For example, the accumulation of PrPSc does not necessarily correlate with disease symptoms in animal models, and the addition of PrPSc to brain tissue that does not also have endogenous or transgenic PrPc will not induce neurodegeneration in this tissue.

Still, the conversion of PrPc to some other form appears to be critical for the disease, so John Collinge, Giovanna Mallucci, and colleagues at the Institute of Neurology in London decided to target PrPc directly. The researchers generated mice transgenic for a prion protein gene that increases the amounts of PrPc produced, but also harboring a transgene for the enzyme Cre recombinase, which shuts down the expression of the PrP transgenes. The beauty of this experimental system is that a neurofilament heavy chain promoter does not allow Cre to be expressed until the mice are 10 to 12 weeks of age, and limits expression to neurons. Thus, the mice overproduce PrPc in both neurons and glia until about 12 weeks of age, after which Cre shuts this expression down in neurons.

The experiment began with the inoculation of mice with scrapie prions at three to four weeks of age. At 12 weeks of age, both PrP and Cre/PrP transgenic animals show pathological evidence of infection, though they still have minimal spongiosis and no behavioral signs of disease. At that point, PrP transgenic mice (lacking the Cre enzyme to shut down PrP in neurons) proceed to develop spongiosis and die around 20 weeks of age.

By contrast, at last count, mice with the Cre gene were still going strong—and asymptomatic—at an average 60 weeks of age. This survival correlates with a shutdown of PrPc in neurons, but not in glia, and a rescue of neurons from degeneration. Surprisingly, however, PrPSc continues to accumulate in these asymptomatic mice. The reason appears to be that glial conversion of PrP is not shut down. What’s more, the little bit of spongiosis that had begun in mice at 12 weeks of age was reversed when Cre expression kicked in.

"Our results…argue against direct neurotoxicity of PrPSc," write the authors. They add that "[I]t appears that the conversion of PrPc to disease-related forms must occur within neurons to be pathogenic, consistent with the possibility that a toxic intermediate is generated within neurons during the conversion process."—Hakon Heimer

Comments

  1. This is an elegant study that shows the importance of studying further the biological role of PrPC, and the intermediates that are formed during the conversion of PrPC to PrPSc. It is interesting to compare these findings to the Alzheimer's field, where little neurotoxicity is associated with Aβ plaques in mouse models, and certain Aβ species may be responsible for Aβ-induced neurotoxicity.

    View all comments by Einar Sigurdsson

  2. A requirement for intraneuronal PrPsc conversion in prion pathogenesis: What questions does this raise about the mechanism of AD pathogenesis?

    The recent report by John Collinge and colleagues provides important new insight into the mechanisms of pathogenesis of prion disease, and raises some interesting questions about the corresponding mechanism for Alzheimer’s disease (AD) pathogenesis. They report that specifically depleting PrPc in neurons at 10-12 weeks reversed the early spongiform degeneration and dramatically prevented neuronal death and clinical disease, even though PrPsc accumulated extracellularly to the same level observed in diseased control animals. These results indicate that prion pathogenesis is intimately associated with the conformational conversion of PrPc to PrPsc in neurons, and that the extracellular accumulation of PrPsc is not toxic per se.

    Do these results have heuristic value as an analogy for AD pathogenesis? AD and prion pathogenesis share some interesting pathogenic features. In AD, the amyloid Aβ peptide accumulates extracellularly, and plaques analogous to the extracellular accumulation of PrPsc and the significance of these deposits have been matters of intense interest (Hardy and Selkoe, 2002). As in prion disease, the accumulation of insoluble extracellular deposits does not correlate well with disease, leading to the development of the concept that diffusible extracellular Aβ oligomers represent intermediates of the primary pathogenic species and not the fibrillar amyloid deposits (Lambert et al., 1998). The results from specifically depleting prion expression in neurons indicate that whatever the pathogenic mechanism, it is intimately associated with the replication process in neurons, and that the exposure to putative extracellular toxins is not sufficient to cause disease. Is this also the case in Alzheimer’s disease? Amyloid Aβ has also been observed to accumulate intracellularly in AD (Gouras et al., 2000; D'Andrea et al., 2001; Takahashi et al., 2002). The accumulation of intracellular Aβ is mechanistically related to prion replication, as it involves the nucleation-dependent accumulation of insoluble APP and amyloidogenic fragments of APP in the endosomal/lysosomal system of cells, followed by the slow conversion of this insoluble residue to Aβ n-42 due to the nonspecific proteolytic degradation of the APP domains outside of the Aβ lattice Yang et al., 1995; Yang et al., 1999; Glabe 2001). The intracellular Aβ model for AD pathogenesis predicts that dystrophic neurites and neuritic plaques arise as a consequence of the intracellular replication of Aβ, and that not all plaque deposits are pathogically significant (Glabe, 2001). It has been known for some time that aged individuals can have large numbers of diffuse amyloid deposits and that some individuals with PS mutations can accumulate impressive deposits of “cotton wool” diffuse plaques without symptoms of dementia, suggesting that diffuse deposits are clinically significant (Hardy and Selkoe, 2002). ApoE isoform is a major risk factor for late-onset AD and ApoE appears to be necessary for the development of neuritic pathology in Tg mouse models (Holtzman et al., 2000). Neuritic pathology is virtually absent in ApoE -/- Tg animals. If neuritic pathology and neuritic plaque accumulation is a reflection of the process of intracellular amyloid accumulation, then ApoE may play a critical role in the nucleation or initiation of this process.

    The data from Collinge and coworkers indicates that extracellular PrPsc deposits derived from non-neuronal sources are not pathologically significant. This illustrates the potential complication of using extracellular amyloid deposition as a surrogate for pathogenesis in animal models of AD. Transplantation studies demonstrated that expression of PrPc in the grafted tissue is necessary for prion pathogenesis even though substantial amounts of graft-derived PrPsc are deposited in the PrPc -/- tissue Brandner et al., 1996). Based on the analogous transplantation of wild-type mouse brain tissue into the brain of human APP Tg mouse models of AD, Mattias Jucker and colleagues demonstrated that human Aβ derived from the surrounding Tg tissue is also deposited in the wild-type mouse graft (Meyer-Luehmann et al., 2003). Although most of the amyloid that was deposited in the wild-type graft was diffuse, an occasional Congophilic plaque was observed with some evidence of surrounding neuritic dystrophy and gliosis. These results have been interpreted as indicating that extracellular amyloid deposition is sufficient for pathogenesis, and that intracellular Aβ is not required for neurodegeneration, but these interpretations are not entirely unambiguous. Unlike the prion experiments where PrP -/- tissue was grafted, the grafted tissue expressed wild-type mouse APP, and it is not clear whether human Aβ can seed the intracellular accumulation of mouse APP and mouse Aβ. This would be analogous to the seeding of new variant CJD by bovine prions. Indeed, antibodies specific for mouse Aβ weakly stain the amyloid deposits in the wild-type graft tissue, indicating some Aβ derived from wild-type mouse APP does accumulate (Meyer-Luehmann et al., 2003). In addition, the mouse AD models do not display the same relatively unambiguous markers of prion neuronal degeneration, like spongiform degeneration, and massive neuronal death. The experiments by Collinge and coworkers also demonstrate that gliosis is not obligately associated with prion pathogenesis. A less ambiguous test of the role of extracellular amyloid in AD pathogenesis and the requirement of intracellular neuronal accumulation of APP and Aβ would be to apply the same experimental strategy used by Collinge and coworkers for prion pathogenesis to Tg mouse models of AD.

    References:

    Brandner S, Isenmann S, Raeber A, Fischer M, Sailer A, Kobayashi Y, Marino S, Weissmann C, Aguzzi A. Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature. 1996 Jan 25;379(6563):339-43. PubMed.

    D'Andrea MR, Nagele RG, Wang HY, Peterson PA, Lee DH. Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease. Histopathology. 2001 Feb;38(2):120-34. PubMed.

    Glabe C. Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer's disease. J Mol Neurosci. 2001 Oct;17(2):137-45. PubMed.

    Gouras GK, Tsai J, Naslund J, Vincent B, Edgar M, Checler F, Greenfield JP, Haroutunian V, Buxbaum JD, Xu H, Greengard P, Relkin NR. Intraneuronal Abeta42 accumulation in human brain. Am J Pathol. 2000 Jan;156(1):15-20. PubMed.

    Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002 Jul 19;297(5580):353-6. PubMed.

    Holtzman DM, Bales KR, Tenkova T, Fagan AM, Parsadanian M, Sartorius LJ, Mackey B, Olney J, McKeel D, Wozniak D, Paul SM. Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2892-7. PubMed.

    Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6448-53. PubMed.

    Meyer-Luehmann M, Stalder M, Herzig MC, Kaeser SA, Kohler E, Pfeifer M, Boncristiano S, Mathews PM, Mercken M, Abramowski D, Staufenbiel M, Jucker M. Extracellular amyloid formation and associated pathology in neural grafts. Nat Neurosci. 2003 Apr;6(4):370-7. PubMed.

    Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H, Beal MF, Xu H, Greengard P, Gouras GK. Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol. 2002 Nov;161(5):1869-79. PubMed.

    Yang AJ, Chandswangbhuvana D, Shu T, Henschen A, Glabe CG. Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abeta1-42. J Biol Chem. 1999 Jul 16;274(29):20650-6. PubMed.

    Yang AJ, Knauer M, Burdick DA, Glabe C. Intracellular A beta 1-42 aggregates stimulate the accumulation of stable, insoluble amyloidogenic fragments of the amyloid precursor protein in transfected cells. J Biol Chem. 1995 Jun 16;270(24):14786-92. PubMed.

    View all comments by Charles Glabe

  3. This study finding that depletion of normal prion protein prevents or reverses the clinical manifestations of Creutzfeldt-Jacob disease is interesting.

    Is it overexpresed as is APP in Alzheimer's disease?

    If the study by Kniazeva, Orman and Terranova finding that PDGF is an inhibitor of PrP is replicated in neuronal tissue, I wonder whether we could look at reasons why PDGF signaling may be inhibited as a cause of prion diseases.

    I had been looking at the fact that chlostridium difficile toxin B inhibits CDC42, and wondered if that would be an alternative to Gleevec for the treatment of Alzheimer's disease. With that in mind, is it too far a stretch to question a C. difficile involvement in the development prion disease? Could inhibition of CDC42 result in reduced inhibition of PrP mRNA by PDGF? Garcia-Lechuz et al. find that extra-intestinal infections caused by C. difficile occurred without concomitant diarrhea. Chang and Rohwer report C. difficile infection as a cause of diarrhoea in adult hampsters used for titrating the scrapie agent.

    References:

    Kniazeva M, Orman R, Terranova VP. Expression of PrP mRNA is regulated by a fragment of MRP8 in human fibroblasts. Biochem Biophys Res Commun. 1997 May 8;234(1):59-63. PubMed.

    García-Lechuz JM, Hernangómez S, Juan RS, Peláez T, Alcalá L, Bouza E. Extra-intestinal infections caused by Clostridium difficile. Clin Microbiol Infect. 2001 Aug;7(8):453-7. PubMed.

    Chang J, Rohwer RG. Clostridium difficile infection in adult hamsters. Lab Anim Sci. 1991 Dec;41(6):548-52. PubMed.

    View all comments by Mary Reid

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Primary Papers

  1. Mallucci G, Dickinson A, Linehan J, Klöhn PC, Brandner S, Collinge J. Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science. 2003 Oct 31;302(5646):871-4. PubMed.

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