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A murine model of a familial prion disease
Clin Lab Med 23 (2003) 175–186
A murine model of a familial prion disease David A. Harris, MD, PhDa,*, Roberto Chiesa, PhDa,1, Bettina Drisaldi, PhDa,2, Elena Quaglioa,1, Antonio Migheli, MD, PhDb, Pedro Piccardo, MDc, Bernardino Ghetti, MDc a
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110, USA b Laboratory of Neuropathology, Department of Neuroscience, University of Turin, 10126 Turin, Italy c Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA
Prion diseases are fatal neurodegenerative disorders of humans and animals that result from the conversion of a normal, cell-surface glycoprotein (PrPC) into a conformationally altered isoform (PrPSc) that is infectious in the absence of nucleic acid [1]. Familial prion diseases, which include 10% of the cases of Creutzfeldt-Jakob disease (CJD) and all cases of GerstmannStra¨ussler syndrome (GSS) and fatal familial insomnia (FFI), are linked to dominantly inherited, germline mutations in the PrP gene on chromosome 20 [2,3]. The mutations are presumed to favor spontaneous conversion of PrPC to PrPSc without the necessity for contact with exogenous infectious agent. Point mutations occur in the C-terminal half of the PrP molecule, and are associated with CJD, GSS, or FFI. Insertional mutations, which are associated with a variable phenotype that can include features of CJD or
This work was supported by grants to D.A.H. from the National Institutes of Health (NIH), the American Health Assistance Foundation, and the Alzheimer’s Association; and to B.G. from the NIH. R.C. was the recipient of fellowships from the Comitato Telethon Fondazione Onlus, and the McDonnell Center for Cellular and Molecular Neurobiology at Washington University. * Corresponding author. E-mail address: [email protected] (D.A. Harris). 1 Present address: Istituto di Ricerche Farmacologiche ‘‘Mario Negri,’’ Milano 20157, Italy. 2 Present address: Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada M5S 3H2. 0272-2712/03/$ - see front matter Ó 2003, Elsevier Science (USA). All rights reserved. doi:10.1016/S0272-2712(02)00069-0
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GSS, consist of one to nine additional copies of a peptide repeat that is normally present in five copies in the N-terminal half of the protein. In humans, this repeat has the sequence P(H/Q)GGG(-/G)WGQ. This article reviews our studies of a murine transgenic (Tg) model of a familial prion disease that is associated with a nine-octapeptide insertion in the PrP gene. The human homolog of this mutation, which is the largest insertion thus far described, was found in two patients (one British and one German) who were afflicted with an illness characterized by progressive dementia and ataxia, and in one autopsied case, by the presence of PrPcontaining amyloid plaques in the cerebellum and basal ganglia [4–6]. The Tg mice that were produced modeled key clinical and neuropathologic features of human familial prion diseases, and, unlike mice that express PrP with a GSS-related point mutation (P101L) [7,8], they spontaneously accumulated mutant proteins that possessed some of the features of PrPSc. Analysis of these mice has provided important insights into the natural history and pathogenesis of familial prion diseases, and has shed new light on the molecular distinction between pathogenic and infectious forms of PrP.
Construction of Tg mice We have constructed mice [designated Tg(PG14)] that harbor a mouse PrP (moPrP) transgene containing a nine-octapeptide insertion homologous to the one found in human patients [9]. We refer to this insertional mutation as PG14, because it results in a protein with a total of 14 repeats that are rich in proline and glycine. The PG14 mutation is one that we have studied extensively in cultured cells [10–15], and it was chosen because we and others [16] have found that PrP molecules with longer insertions have more prominent PrPSc-like biochemical properties. For control purposes, we also have produced mice [designated Tg(WT)] that express a wild-type moPrP transgene. Both wild-type and PG14 molecules carry an epitope tag for the monoclonal antibody 3F4 [17], which makes it possible to distinguish transgene-encoded PrP from endogenous moPrP. This epitope tag, which consists of substitution of methionine residues at positions 108 and 111 of moPrP, does not, by itself, affect the properties of the PrP molecule [18]. Wild-type and PG14 PrP cDNAs were cloned in the Tg vector MoPrP.Xho, which contains the promoter and intron 1 of the moPrP gene, and has been shown to produce expression of foreign genes in a tissue and developmental pattern similar to that of endogenous PrP, with the exception that expression is absent in cerebellar Purkinje cells [19,20]. Tg founders produced on a C57BL/6J CBA/J background were bred back to this line, and also were crossed into a Prn-p0/0 line [21] in which both copies of the endogenous PrP gene had been disrupted.
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Tg(PG14) mice develop neurologic symptoms Tg(PG14) mice from the A2 and A3 lines (both of which express mutant PrP at levels similar to that of endogenous PrP) develop a progressive and ultimately fatal neurologic disorder characterized by ataxia, kyphosis, footclasp reflex, waddling gait, difficulty righting, and weight loss [9,22]. In contrast, Tg(WT) mice that express wild-type PrP at even higher levels (three to four times that of endogenous PrP) remain healthy. We observed that breeding the transgene array to homozygosity dramatically accelerated the onset of disease (from 235 10 to 68 9 days of age), and shortened its duration (from 154 14 to 49 11 days). This effect is most likely attributable to the twofold higher expression of PG14 PrP in homozygous compared with heterozygous mice. This explanation is consistent with our finding that the Tg(PG14) B and C lines, which express low levels of the mutant protein (15% of the endogenous PrP level), do not develop a neurologic disorder within the life span of the animals. Taken together, our results indicate that overexpression of wild-type PrP does not produce neurologic dysfunction, and that development of the disease in Tg(PG14) mice is related to the expression level of the mutant protein. We observed relatively little effect of coexpression of endogenous wild-type PrP on the characteristics of the illness.
Tg(PG14) mice display neuropathologic abnormalities Several pathologic changes were observed in the A2 and A3 lines of Tg(PG14) mice [9,22]. The most obvious was a massive degeneration of cerebellar granule cells, which begins by 30 days of age in Tg(PG14þ/þ) mice, and eventually results in severe atrophy of the cerebellum (Fig. 1A–C). Several features indicate that granule cell loss occurs by an apoptotic mechanism. These include the presence of numerous pyknotic and fragmented granule cell nuclei (Fig. 1D), positive staining of degenerating neurons both by in situ end labeling (ISEL) of DNA (Fig. 1E–G) and by an antibody to activated caspase-3, and the presence in cerebellar DNA preparations of a 200-base pair ladder indicative of internucleosomal cleavage. We are currently determining by ISEL staining whether neurons, in addition to cerebellar granule cells, undergo apoptosis in Tg(PG14) mice; preliminary evidence indicates that some sets of hippocampal neurons also may be affected. Granule cell loss also is seen in a number of cases of human prion diseases [23]. Although it is generally agreed that neuronal loss is a cardinal feature of prion diseases, the role of apoptosis in this process has received limited attention [24–27]. Tg(PG14) mice thus provide a particularly clear-cut demonstration of the role of neuronal apoptosis in a prion disease, and allow for the exploration of several hypotheses about the underlying cellular and molecular mechanisms.
Fig. 1. Neuropathologic changes in the cerebella of Tg(PG14þ/þ) mice. Hematoxylin-eosin stained sections showing the cerebellar cortex of mice of 22 days (A), 100 days (B, D), and 183 days (C) of age. M, molecular layer; PC, Purkinje cell layer; G, granule cell layer. Note the dramatic decrease in the number of granule cells with age. Arrowheads in (D) indicate pyknotic nuclei. ISEL-stained sections showing positively stained cells (brown) in the granule cell layer from mice of 31 days (E), 53 days (F), and 181 days (G) of age. PrP immunostaining of cerebellar cortex from mice of 22 days (H), 100 days (I), and 181 days (J) of age. Note the small, punctate deposits of PrP. Scale bars are 50 lm (A–C), 10 lm (D), 13 lm (E–G), and 32 lm (H–J). (See also Color Plate 15). (From Chiesa R, Drisaldi B, Quaglio E, et al. Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation. Proc Natl Acad Sci USA 2000;97:5577; with permission.)
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A second abnormal feature in Tg(PG14) mice was the presence of punctate, ‘‘synaptic-like’’ deposits of PrP that labeled with antibody 3F4 following treatment of brain sections with guanidine thiocyanate and hydrolytic autoclaving (Fig. 1H–J). These deposits were most prominent in the cerebellum, hippocampal formation, and olfactory bulb, and were present to a lesser extent in the neocortex and inferior colliculus. A third finding was astrocytic gliosis, which was observed in the cerebellar cortex (particularly the molecular layer, which displayed markedly hypertrophied Bergmann glial fibers), the hippocampus, and the neocortex. PrP deposition and gliosis began at 30 to 40 days of age in Tg(PG14þ/þ) mice, and increased as the illness progressed. Neither thioflavin-S fluorescent plaques, nor obvious spongiosis were observed at any time. No pathologic changes were seen in Tg(WT) or non-Tg mice. PG14 PrP shares some biochemical properties of PrPSc PrPSc can be recognized by several distinctive biochemical properties, including detergent insolubility, protease resistance, and resistance of the glycosylphosphatidylinositol (GPI) anchor to cleavage by the enzyme phosphatidylinositol-specific phospholipase C (PIPLC). We have found that mutant PrP from the brains of Tg(PG14) mice displays each of these characteristics [9]. We determined the amount of PrP that is detergent insoluble by subjecting Triton-deoxycholate extracts of brain to ultracentrifugation under conditions that sediment PrPSc but leave PrPC in the supernatant. PG14 PrP from all Tg(PG14) lines was partially detergent insoluble, whereas wild-type PrP from Tg(WT) mice was completely soluble. To assess protease resistance, we digested detergent extracts of brain with 1 to 5 lg/mL of proteinase K (PK) for 30 min at 37C. We found that, under these conditions, PG14 PrP from all Tg lines yielded a protease-resistant core fragment of 27 to 30 kDa (PrP 27-30) (Fig. 2B, C). In contrast, moPrP from all four Tg(WT) lines was completely degraded at PK concentrations as low as 0.5 lg/mL (Fig. 2A). Mapping of the immunoreactive epitopes present in PrP 27-30 from Tg(PG14) mice suggested that this fragment is produced by cleavage between the end of the octapeptide repeats and amino acid 94, the same region in which authentic PrPSc is cut. To assay PIPLC resistance, suspensions of brain microsomal membranes were treated with phospholipase, and the amount of PrP released into the medium was determined by immunoblotting pellet and supernatant fractions obtained after sedimenting the membranes by centrifugation. Whereas approximately 50% of the wild-type PrP from Tg(WT) mice was releasable by PIPLC, none of the PG14 PrP from Tg(PG14) mice was releasable. PG14 PrP also was found to be relatively PIPLC resistant in a second assay in which removal of the GPI anchor was measured by Triton X-114 phase partitioning. Mutant PrP in the brains of Tg(PG14) mice differs from authentic PrPSc in an important respect, namely its considerably lower level of protease
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resistance. PrPSc found in the brains of humans and animals with familial, infectious, or sporadic prion diseases will typically yield a PrP 27-30 fragment after treatment with 20 to 100 lg/mL of PK for 0.5 to 2 hours at 37C. The PK concentration that we have used to digest PG14 PrP from our Tg mice is 10 to 100 times lower. This biochemical difference raises the question of whether PG14 PrP is authentic PrPSc, an issue that is discussed below. PG14 PrP and its PrPSc-like isoform accumulate throughout life Detergent-insoluble and protease-resistant PG14 PrP was synthesized in the brains of Tg mice during the first week of life, well before the animals developed clinical symptoms or neuropathologic changes [22]. Moreover, the amount of detergent-insoluble and protease-resistant PrP increased dramatically with age, with levels in the oldest, terminally ill animals that were 20-fold to 80-fold higher than in newborn mice (Fig. 3). PrPSc-like protein accumulated more rapidly and to higher levels in Tg(PG14þ/þ) mice than in Tg(PG14þ/) mice, correlating with the accelerated disease progression in the homozygous animals. These results indicate that mutant PrP is converted continuously to the PrPSc state throughout life, but that clinical symptoms and neuropathologic lesions do not ensue until the amount of PrPSc reaches a critical threshold level. If the same is true in human patients, this would explain why familial prion diseases do not manifest themselves clinically until adulthood, even though mutant PrP is synthesized from before birth [28]. Accumulation of the PrPSc isoform paralleled an increase in the total amount of PG14 PrP in Tg(PG14) mice [22]. As Tg(PG14) mice aged, there was a nearly 10-fold increase in the total amount of mutant PrP in their brains. This phenomenon was specific for the mutant form of the protein, because the amount of wild-type PrP in the brains of Tg(WT) and non-Tg CD1 mice varied by less than twofold over the lifetime of the animals. To address the possibility that accumulation of PG14 PrP was due to increased b Fig. 2. PG14 PrP in the brains of Tg mice is protease resistant. Detergent lysates of brain from Tg mice and Syrian hamster were incubated with the indicated amounts of PK for 30 minutes at 37C. Digestion was terminated by addition of phenylmethylsulforyl fluoride, and methanolprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using antibody 3F4. 200 lg (A, B) or 800 lg (C) of initial protein was subjected to digestion. The lanes containing undigested samples (0 lg/mL PK) represent 50 lg (A, B) or 200 lg (C) of protein. Mice were of the following ages and clinical status at the time of sacrifice: E1 through E4, 203 days old (all healthy); A1, 319 days old (severely symptomatic); A2, 71 days old (healthy); A3, 205 days old (mildly symptomatic); B, 169 days old (healthy); C, 226 days old (healthy). (From Chiesa R, Piccardo P, Ghetti B, et al. Neurological illness in transgenic mice expressing a prion protein with an insertional mutation. Neuron 1998;21:1345; with permission.)
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Fig. 3. The amount of detergent-insoluble and protease-resistant PG14 PrP increases with age in the brains of Tg(PG14) mice. (A) Brain lysates from Tg(PG14þ/þ) mice of the indicated ages were subjected to ultracentrifugation, and PrP in the supernatants (S lanes) and pellets (P lanes) was analyzed by Western blotting. (B) The amount of PG14 PrP in the pellet fraction after ultracentrifugation (see A) was quantitated by densitometric analysis of Western blots of samples from Tg(PG14þ/) and Tg(PG14þ/þ) mice. Each bar represents the mean SEM of four to eight replicate analyses of samples from four to seven brains. (C) Brain lysates from Tg(PG14þ/þ) mice of the indicated ages were incubated with 0 to 3 lg of PK for 30 min at 37C, and PrP was visualized by Western blotting. The undigested samples (0 lg/mL PK) represent 50 lg of protein, and the other samples represent 200 lg of protein. The protease-resistant fragment (PrP 27-30) migrates between 27 and 30 kDa. (D) The amount of PrP 27-30 that was produced by digestion with 2 lg/mL of PK (see C) was quantitated by densitometric analysis of Western blots of samples from Tg(PG14þ/) and Tg(PG14þ/þ) mice. Each bar represents the mean SEM of four to eight replicate analyses of samples from four to seven brains. (From Chiesa R, Drisaldi B, Quaglio E, et al. Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation. Proc Natl Acad Sci USA 2000;97:5575; with permission.)
gene transcription, we performed Northern blots to assay PrP mRNA, and found that there was no significant change in the amount of transgenically encoded PrP mRNA during postnatal development of either Tg(PG14) or Tg(WT) mice. These results imply that PG14 PrP is degraded or cleared more slowly than is wild-type PrP. PrPSc-like molecules are widely distributed in Tg(PG14) mice To assess the neuroanatomic distribution of the PrPSc-like form of PG14 PrP, we carried out histoblots of cryostat sections of brain [22]. Proteaseresistant PrP was found throughout the brains of both preclinical and
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terminally ill Tg(PG14) mice, with particular concentrations in the medial caudate putamen, septum, corpus callosum, anterior commissure, ventral thalamus, globus pallidus, and hippocampus. Protease-resistant and detergent-insoluble PrP also was detected by Western blot analysis of individually dissected brain regions [22]. The widespread anatomical distribution of the PrPSc revealed by these methods contrasts with the more restricted distribution of punctate PrP deposits seen by immunohistochemistry, raising the possibility that PrPSc may be more aggregated in certain regions such as the cerebellum. Taken together, these results indicate that many neuronal populations are capable of converting PG14 PrP to a PrPSclike state. Surprisingly, we have also found a detergent-insoluble and proteaseresistant form of PG14 PrP in a number of the peripheral tissues in which PrP is normally expressed (although at lower levels than in the brain), including skeletal muscle, heart, kidney, and testis [22]. To see whether accumulation of this PrPSc-like protein had pathologic consequences, we carried out histologic and functional analyses of peripheral tissues, and discovered that Tg(PG14) mice display a progressive, primary skeletal myopathy characterized by the presence of central nuclei, necrotic and regenerating myofibers, and variable fiber size [29]. Interestingly, cardiac tissue was histologically normal, as was cardiac function assayed by echocardiography. Because expression levels of PG14 PrP are similar in skeletal and cardiac muscle, these data suggest that cell types differ in their intrinsic susceptibility to pathologic effects of mutant PrP. Given the widespread distribution of protease-resistant and detergent-insoluble PrP throughout the central nervous system and periphery of Tg(PG14) mice, we concluded that structural features of the mutant protein itself, as opposed to cellular factors, play a primary role in its conversion to a PrPSc-like state.
Is PG14 PrP from Tg(PG14) mice infectious? The question of whether PG14 PrP from Tg(PG14) mice is infectious is a crucial one; an affirmative answer would prove that the mutant protein represents authentic PrPSc and would provide another demonstration of the protein-only theory of infectivity. Therefore, we inoculated brain homogenates from clinically ill Tg(PG14) mice of the A2 and A3 lines into several different hosts, including (1) low-expressing Tg(PG14-C) mice that do not become sick spontaneously, (2) Tg(WT) mice, and (3) non-Tg CD1 mice. None of the recipient mice became ill, and none displayed protease-resistant PrP in their brains by Western blotting during an observation period of 500 to 650 days postinoculation, at the end of which time significant numbers of animals were dying of intercurrent illness. Our conclusion from these data was that PG14 PrP produced in Tg mice either has a low titer of infectivity, or is not infectious at all (Chiesa R, Harris D, unpublished results, 2002).
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Interestingly, when Tg(PG14) mice were inoculated with a mouseadapted Rocky Mountain Laboratory strain of scrapie, they accumulated a form of PG14 PrP that was resistant to high concentrations of PK, and was infectious upon serial passage (Chiesa R, Harris D, unpublished results 2002). Thus, two forms of mutant PrP with identical primary sequences, but different degrees of protease resistance and infectivity are produced in Tg(PG14) mice, depending on whether the protein accumulates spontaneously or is ‘‘seeded’’ by exogenous prions. Structural analysis of these two isoforms will provide important insights into the molecular features of PrP that are essential for pathogenicity and the modifications associated with acquisition of infectivity. These studies highlight what has recently become a crucial question in the prion field—namely, whether PrPSc itself is the pathogenic form of PrP. Increasing evidence now suggests that although PrPSc is the infectious form of the protein, it is not the form that ultimately kills neurons [30]. We hypothesize that the weakly PK-resistant form of PrP that accumulates in Tg(PG14) mice is an example of a pathogenic but noninfectious form of PrP that may be present during the course of all prion diseases.
Summary We have produced a mouse model of a familial prion disorder by introduction of a transgene that encodes the moPrP homolog of a nineoctapeptide insertional mutant associated with an inherited form of CJD in humans. These mice develop progressive neurologic symptoms, display neuropathologic changes, and accumulate a form of mutant PrP in their brains and peripheral tissues that displays some of the biochemical properties of PrPSc. These mice have been extremely valuable for analyzing the cellular and biochemical mechanisms involved in inherited prion disorders and correlating the appearance of the PrPSc-like form with clinical and neuropathologic findings. Because the mutant protein in the mice is highly neurotoxic but appears to lack infectivity, further analysis of its properties promises to shed new light on the molecular distinction between pathogenic and infectious forms of PrP.
References [1] Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci 2001;24:519–50. [2] Gambetti P, Petersen RB, Parchi P, et al. Inherited prion diseases. In: Prusiner SB, editor. Prion biology and diseases. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1999. p. 509–83. [3] Young K, Piccardo P, Dlouhy S, et al. The human genetic prion diseases. In: Harris DA, editor. Prions: molecular and cellular biology. Wymondham (UK): Horizon Scientific Press; 1999. p. 139–75.
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[4] Duchen LW, Poulter M, Harding AE. Dementia associated with a 216 base pair insertion in the prion protein gene: clinical and neuropathological features. Brain 1993;116: 555–67. [5] Krasemann S, Zerr I, Weber T, et al. Prion disease associated with a novel nine octapeptide repeat insertion in the PRNP gene. Mol Brain Res 1995;34:173–6. [6] Owen F, Poulter M, Collinge J, et al. A dementing illness associated with a novel insertion in the prion protein gene. Mol Brain Res 1992;13:155–7. [7] Hsiao KK, Scott M, Foster D, et al. Spontaneous neurodegeneration in transgenic mice with mutant prion protein. Science 1990;250:1587–90. [8] Telling GC, Haga T, Torchia M, et al. Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice. Genes Dev 1996;10:1736–50. [9] Chiesa R, Piccardo P, Ghetti B, et al. Neurological illness in transgenic mice expressing a prion protein with an insertional mutation. Neuron 1998;21:1339–51. [10] Chiesa R, Harris DA. Nerve growth factor-induced differentiation does not alter the biochemical properties of a mutant prion protein expressed in PC12 cells. J Neurochem 2000;75:72–80. [11] Daude N, Lehmann S, Harris DA. Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells. J Biol Chem 1997; 272:11604–12. [12] Lehmann S, Harris DA. Mutant and infectious prion proteins display common biochemical properties in cultured cells. J Biol Chem 1996;271:1633–7. [13] Lehmann S, Harris DA. A mutant prion protein displays an aberrant membrane association when expressed in cultured cells. J Biol Chem 1995;270:24589–97. [14] Lehmann S, Harris DA. Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform. Proc Natl Acad Sci USA 1996;93:5610–4. [15] Narwa R, Harris DA. Prion proteins carrying pathogenic mutations are resistant to phospholipase cleavage of their glycolipid anchors. Biochemistry 1999;38:8770–7. [16] Priola SA, Chesebro B. Abnormal properties of prion protein with insertional mutations in different cell types. J Biol Chem 1998;273:11980–5. [17] Bolton DC, Seligman SJ, Bablanian G, et al. Molecular location of a species-specific epitope on the hamster scrapie agent protein. J Virol 1991;65:3667–75. [18] Scott M, Groth D, Foster D, et al. Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 1993;73:979–88. [19] Borchelt DR, Davis J, Fischer M, et al. A vector for expressing foreign genes in the brains and hearts of transgenic mice. Genet Anal Biomol Eng 1996;13:159–63. [20] Fischer M, Ru¨licke T, Raeber A, et al. Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J 1996;15:1255–64. [21] Bu¨eler H, Fischer M, Lang Y, et al. Normal development and behavior of mice lacking the neuronal cell-surface PrP protein. Nature 1992;356:577–82. [22] Chiesa R, Drisaldi B, Quaglio E, et al. Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation. Proc Natl Acad Sci USA 2000;97:5574–9. [23] Parchi P, Gambetti P, Piccardo P, et al. Human prion diseases. In: Kirkham N, Lemoine NR, editors. Progress in pathology 4. Edinburgh: Churchill Livingstone; 1998. p. 39–77. [24] Dorandeu A, Wingertsmann L, Chre´tien F, et al. Neuronal apoptosis in fatal familial insomnia. Brain Pathol 1998;8:531–7. [25] Giese A, Groschup MH, Hess B, et al. Neuronal cell death in scrapie-infected mice is due to apoptosis. Brain Pathol 1995;5:213–21. [26] Gray F, Chre´tien F, Adle-Biassette H, et al. Neuronal apoptosis in Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 1999;58:321–8. [27] Lucassen PJ, Williams A, Chung WCJ, et al. Detection of apoptosis in murine scrapie. Neurosci Lett 1995;198:185–8.
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[28] Manson J, West JD, Thomson V, et al. The prion protein gene: a role in mouse embryogenesis? Development 1992;115:117–22. [29] Chiesa R, Pestronk A, Schmidt RE, et al. Primary myopathy and accumulation of PrPSclike molecules in peripheral tissues of transgenic mice expressing a prion protein insertional mutation. Neurobiol Dis 2001;8:279–88. [30] Chiesa R, Harris DA. Prion diseases: what is the neurotoxic molecule? Neurobiol Dis 2001;8:743–63.
A murine model of a familial prion disease David A. Harris, MD, PhDa,*, Roberto Chiesa, PhDa,1, Bettina Drisaldi, PhDa,2, Elena Quaglioa,1, Antonio Migheli, MD, PhDb, Pedro Piccardo, MDc, Bernardino Ghetti, MDc a
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110, USA b Laboratory of Neuropathology, Department of Neuroscience, University of Turin, 10126 Turin, Italy c Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA
Prion diseases are fatal neurodegenerative disorders of humans and animals that result from the conversion of a normal, cell-surface glycoprotein (PrPC) into a conformationally altered isoform (PrPSc) that is infectious in the absence of nucleic acid [1]. Familial prion diseases, which include 10% of the cases of Creutzfeldt-Jakob disease (CJD) and all cases of GerstmannStra¨ussler syndrome (GSS) and fatal familial insomnia (FFI), are linked to dominantly inherited, germline mutations in the PrP gene on chromosome 20 [2,3]. The mutations are presumed to favor spontaneous conversion of PrPC to PrPSc without the necessity for contact with exogenous infectious agent. Point mutations occur in the C-terminal half of the PrP molecule, and are associated with CJD, GSS, or FFI. Insertional mutations, which are associated with a variable phenotype that can include features of CJD or
This work was supported by grants to D.A.H. from the National Institutes of Health (NIH), the American Health Assistance Foundation, and the Alzheimer’s Association; and to B.G. from the NIH. R.C. was the recipient of fellowships from the Comitato Telethon Fondazione Onlus, and the McDonnell Center for Cellular and Molecular Neurobiology at Washington University. * Corresponding author. E-mail address: [email protected] (D.A. Harris). 1 Present address: Istituto di Ricerche Farmacologiche ‘‘Mario Negri,’’ Milano 20157, Italy. 2 Present address: Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada M5S 3H2. 0272-2712/03/$ - see front matter Ó 2003, Elsevier Science (USA). All rights reserved. doi:10.1016/S0272-2712(02)00069-0
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GSS, consist of one to nine additional copies of a peptide repeat that is normally present in five copies in the N-terminal half of the protein. In humans, this repeat has the sequence P(H/Q)GGG(-/G)WGQ. This article reviews our studies of a murine transgenic (Tg) model of a familial prion disease that is associated with a nine-octapeptide insertion in the PrP gene. The human homolog of this mutation, which is the largest insertion thus far described, was found in two patients (one British and one German) who were afflicted with an illness characterized by progressive dementia and ataxia, and in one autopsied case, by the presence of PrPcontaining amyloid plaques in the cerebellum and basal ganglia [4–6]. The Tg mice that were produced modeled key clinical and neuropathologic features of human familial prion diseases, and, unlike mice that express PrP with a GSS-related point mutation (P101L) [7,8], they spontaneously accumulated mutant proteins that possessed some of the features of PrPSc. Analysis of these mice has provided important insights into the natural history and pathogenesis of familial prion diseases, and has shed new light on the molecular distinction between pathogenic and infectious forms of PrP.
Construction of Tg mice We have constructed mice [designated Tg(PG14)] that harbor a mouse PrP (moPrP) transgene containing a nine-octapeptide insertion homologous to the one found in human patients [9]. We refer to this insertional mutation as PG14, because it results in a protein with a total of 14 repeats that are rich in proline and glycine. The PG14 mutation is one that we have studied extensively in cultured cells [10–15], and it was chosen because we and others [16] have found that PrP molecules with longer insertions have more prominent PrPSc-like biochemical properties. For control purposes, we also have produced mice [designated Tg(WT)] that express a wild-type moPrP transgene. Both wild-type and PG14 molecules carry an epitope tag for the monoclonal antibody 3F4 [17], which makes it possible to distinguish transgene-encoded PrP from endogenous moPrP. This epitope tag, which consists of substitution of methionine residues at positions 108 and 111 of moPrP, does not, by itself, affect the properties of the PrP molecule [18]. Wild-type and PG14 PrP cDNAs were cloned in the Tg vector MoPrP.Xho, which contains the promoter and intron 1 of the moPrP gene, and has been shown to produce expression of foreign genes in a tissue and developmental pattern similar to that of endogenous PrP, with the exception that expression is absent in cerebellar Purkinje cells [19,20]. Tg founders produced on a C57BL/6J CBA/J background were bred back to this line, and also were crossed into a Prn-p0/0 line [21] in which both copies of the endogenous PrP gene had been disrupted.
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Tg(PG14) mice develop neurologic symptoms Tg(PG14) mice from the A2 and A3 lines (both of which express mutant PrP at levels similar to that of endogenous PrP) develop a progressive and ultimately fatal neurologic disorder characterized by ataxia, kyphosis, footclasp reflex, waddling gait, difficulty righting, and weight loss [9,22]. In contrast, Tg(WT) mice that express wild-type PrP at even higher levels (three to four times that of endogenous PrP) remain healthy. We observed that breeding the transgene array to homozygosity dramatically accelerated the onset of disease (from 235 10 to 68 9 days of age), and shortened its duration (from 154 14 to 49 11 days). This effect is most likely attributable to the twofold higher expression of PG14 PrP in homozygous compared with heterozygous mice. This explanation is consistent with our finding that the Tg(PG14) B and C lines, which express low levels of the mutant protein (15% of the endogenous PrP level), do not develop a neurologic disorder within the life span of the animals. Taken together, our results indicate that overexpression of wild-type PrP does not produce neurologic dysfunction, and that development of the disease in Tg(PG14) mice is related to the expression level of the mutant protein. We observed relatively little effect of coexpression of endogenous wild-type PrP on the characteristics of the illness.
Tg(PG14) mice display neuropathologic abnormalities Several pathologic changes were observed in the A2 and A3 lines of Tg(PG14) mice [9,22]. The most obvious was a massive degeneration of cerebellar granule cells, which begins by 30 days of age in Tg(PG14þ/þ) mice, and eventually results in severe atrophy of the cerebellum (Fig. 1A–C). Several features indicate that granule cell loss occurs by an apoptotic mechanism. These include the presence of numerous pyknotic and fragmented granule cell nuclei (Fig. 1D), positive staining of degenerating neurons both by in situ end labeling (ISEL) of DNA (Fig. 1E–G) and by an antibody to activated caspase-3, and the presence in cerebellar DNA preparations of a 200-base pair ladder indicative of internucleosomal cleavage. We are currently determining by ISEL staining whether neurons, in addition to cerebellar granule cells, undergo apoptosis in Tg(PG14) mice; preliminary evidence indicates that some sets of hippocampal neurons also may be affected. Granule cell loss also is seen in a number of cases of human prion diseases [23]. Although it is generally agreed that neuronal loss is a cardinal feature of prion diseases, the role of apoptosis in this process has received limited attention [24–27]. Tg(PG14) mice thus provide a particularly clear-cut demonstration of the role of neuronal apoptosis in a prion disease, and allow for the exploration of several hypotheses about the underlying cellular and molecular mechanisms.
Fig. 1. Neuropathologic changes in the cerebella of Tg(PG14þ/þ) mice. Hematoxylin-eosin stained sections showing the cerebellar cortex of mice of 22 days (A), 100 days (B, D), and 183 days (C) of age. M, molecular layer; PC, Purkinje cell layer; G, granule cell layer. Note the dramatic decrease in the number of granule cells with age. Arrowheads in (D) indicate pyknotic nuclei. ISEL-stained sections showing positively stained cells (brown) in the granule cell layer from mice of 31 days (E), 53 days (F), and 181 days (G) of age. PrP immunostaining of cerebellar cortex from mice of 22 days (H), 100 days (I), and 181 days (J) of age. Note the small, punctate deposits of PrP. Scale bars are 50 lm (A–C), 10 lm (D), 13 lm (E–G), and 32 lm (H–J). (See also Color Plate 15). (From Chiesa R, Drisaldi B, Quaglio E, et al. Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation. Proc Natl Acad Sci USA 2000;97:5577; with permission.)
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A second abnormal feature in Tg(PG14) mice was the presence of punctate, ‘‘synaptic-like’’ deposits of PrP that labeled with antibody 3F4 following treatment of brain sections with guanidine thiocyanate and hydrolytic autoclaving (Fig. 1H–J). These deposits were most prominent in the cerebellum, hippocampal formation, and olfactory bulb, and were present to a lesser extent in the neocortex and inferior colliculus. A third finding was astrocytic gliosis, which was observed in the cerebellar cortex (particularly the molecular layer, which displayed markedly hypertrophied Bergmann glial fibers), the hippocampus, and the neocortex. PrP deposition and gliosis began at 30 to 40 days of age in Tg(PG14þ/þ) mice, and increased as the illness progressed. Neither thioflavin-S fluorescent plaques, nor obvious spongiosis were observed at any time. No pathologic changes were seen in Tg(WT) or non-Tg mice. PG14 PrP shares some biochemical properties of PrPSc PrPSc can be recognized by several distinctive biochemical properties, including detergent insolubility, protease resistance, and resistance of the glycosylphosphatidylinositol (GPI) anchor to cleavage by the enzyme phosphatidylinositol-specific phospholipase C (PIPLC). We have found that mutant PrP from the brains of Tg(PG14) mice displays each of these characteristics [9]. We determined the amount of PrP that is detergent insoluble by subjecting Triton-deoxycholate extracts of brain to ultracentrifugation under conditions that sediment PrPSc but leave PrPC in the supernatant. PG14 PrP from all Tg(PG14) lines was partially detergent insoluble, whereas wild-type PrP from Tg(WT) mice was completely soluble. To assess protease resistance, we digested detergent extracts of brain with 1 to 5 lg/mL of proteinase K (PK) for 30 min at 37C. We found that, under these conditions, PG14 PrP from all Tg lines yielded a protease-resistant core fragment of 27 to 30 kDa (PrP 27-30) (Fig. 2B, C). In contrast, moPrP from all four Tg(WT) lines was completely degraded at PK concentrations as low as 0.5 lg/mL (Fig. 2A). Mapping of the immunoreactive epitopes present in PrP 27-30 from Tg(PG14) mice suggested that this fragment is produced by cleavage between the end of the octapeptide repeats and amino acid 94, the same region in which authentic PrPSc is cut. To assay PIPLC resistance, suspensions of brain microsomal membranes were treated with phospholipase, and the amount of PrP released into the medium was determined by immunoblotting pellet and supernatant fractions obtained after sedimenting the membranes by centrifugation. Whereas approximately 50% of the wild-type PrP from Tg(WT) mice was releasable by PIPLC, none of the PG14 PrP from Tg(PG14) mice was releasable. PG14 PrP also was found to be relatively PIPLC resistant in a second assay in which removal of the GPI anchor was measured by Triton X-114 phase partitioning. Mutant PrP in the brains of Tg(PG14) mice differs from authentic PrPSc in an important respect, namely its considerably lower level of protease
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resistance. PrPSc found in the brains of humans and animals with familial, infectious, or sporadic prion diseases will typically yield a PrP 27-30 fragment after treatment with 20 to 100 lg/mL of PK for 0.5 to 2 hours at 37C. The PK concentration that we have used to digest PG14 PrP from our Tg mice is 10 to 100 times lower. This biochemical difference raises the question of whether PG14 PrP is authentic PrPSc, an issue that is discussed below. PG14 PrP and its PrPSc-like isoform accumulate throughout life Detergent-insoluble and protease-resistant PG14 PrP was synthesized in the brains of Tg mice during the first week of life, well before the animals developed clinical symptoms or neuropathologic changes [22]. Moreover, the amount of detergent-insoluble and protease-resistant PrP increased dramatically with age, with levels in the oldest, terminally ill animals that were 20-fold to 80-fold higher than in newborn mice (Fig. 3). PrPSc-like protein accumulated more rapidly and to higher levels in Tg(PG14þ/þ) mice than in Tg(PG14þ/) mice, correlating with the accelerated disease progression in the homozygous animals. These results indicate that mutant PrP is converted continuously to the PrPSc state throughout life, but that clinical symptoms and neuropathologic lesions do not ensue until the amount of PrPSc reaches a critical threshold level. If the same is true in human patients, this would explain why familial prion diseases do not manifest themselves clinically until adulthood, even though mutant PrP is synthesized from before birth [28]. Accumulation of the PrPSc isoform paralleled an increase in the total amount of PG14 PrP in Tg(PG14) mice [22]. As Tg(PG14) mice aged, there was a nearly 10-fold increase in the total amount of mutant PrP in their brains. This phenomenon was specific for the mutant form of the protein, because the amount of wild-type PrP in the brains of Tg(WT) and non-Tg CD1 mice varied by less than twofold over the lifetime of the animals. To address the possibility that accumulation of PG14 PrP was due to increased b Fig. 2. PG14 PrP in the brains of Tg mice is protease resistant. Detergent lysates of brain from Tg mice and Syrian hamster were incubated with the indicated amounts of PK for 30 minutes at 37C. Digestion was terminated by addition of phenylmethylsulforyl fluoride, and methanolprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using antibody 3F4. 200 lg (A, B) or 800 lg (C) of initial protein was subjected to digestion. The lanes containing undigested samples (0 lg/mL PK) represent 50 lg (A, B) or 200 lg (C) of protein. Mice were of the following ages and clinical status at the time of sacrifice: E1 through E4, 203 days old (all healthy); A1, 319 days old (severely symptomatic); A2, 71 days old (healthy); A3, 205 days old (mildly symptomatic); B, 169 days old (healthy); C, 226 days old (healthy). (From Chiesa R, Piccardo P, Ghetti B, et al. Neurological illness in transgenic mice expressing a prion protein with an insertional mutation. Neuron 1998;21:1345; with permission.)
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Fig. 3. The amount of detergent-insoluble and protease-resistant PG14 PrP increases with age in the brains of Tg(PG14) mice. (A) Brain lysates from Tg(PG14þ/þ) mice of the indicated ages were subjected to ultracentrifugation, and PrP in the supernatants (S lanes) and pellets (P lanes) was analyzed by Western blotting. (B) The amount of PG14 PrP in the pellet fraction after ultracentrifugation (see A) was quantitated by densitometric analysis of Western blots of samples from Tg(PG14þ/) and Tg(PG14þ/þ) mice. Each bar represents the mean SEM of four to eight replicate analyses of samples from four to seven brains. (C) Brain lysates from Tg(PG14þ/þ) mice of the indicated ages were incubated with 0 to 3 lg of PK for 30 min at 37C, and PrP was visualized by Western blotting. The undigested samples (0 lg/mL PK) represent 50 lg of protein, and the other samples represent 200 lg of protein. The protease-resistant fragment (PrP 27-30) migrates between 27 and 30 kDa. (D) The amount of PrP 27-30 that was produced by digestion with 2 lg/mL of PK (see C) was quantitated by densitometric analysis of Western blots of samples from Tg(PG14þ/) and Tg(PG14þ/þ) mice. Each bar represents the mean SEM of four to eight replicate analyses of samples from four to seven brains. (From Chiesa R, Drisaldi B, Quaglio E, et al. Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation. Proc Natl Acad Sci USA 2000;97:5575; with permission.)
gene transcription, we performed Northern blots to assay PrP mRNA, and found that there was no significant change in the amount of transgenically encoded PrP mRNA during postnatal development of either Tg(PG14) or Tg(WT) mice. These results imply that PG14 PrP is degraded or cleared more slowly than is wild-type PrP. PrPSc-like molecules are widely distributed in Tg(PG14) mice To assess the neuroanatomic distribution of the PrPSc-like form of PG14 PrP, we carried out histoblots of cryostat sections of brain [22]. Proteaseresistant PrP was found throughout the brains of both preclinical and
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terminally ill Tg(PG14) mice, with particular concentrations in the medial caudate putamen, septum, corpus callosum, anterior commissure, ventral thalamus, globus pallidus, and hippocampus. Protease-resistant and detergent-insoluble PrP also was detected by Western blot analysis of individually dissected brain regions [22]. The widespread anatomical distribution of the PrPSc revealed by these methods contrasts with the more restricted distribution of punctate PrP deposits seen by immunohistochemistry, raising the possibility that PrPSc may be more aggregated in certain regions such as the cerebellum. Taken together, these results indicate that many neuronal populations are capable of converting PG14 PrP to a PrPSclike state. Surprisingly, we have also found a detergent-insoluble and proteaseresistant form of PG14 PrP in a number of the peripheral tissues in which PrP is normally expressed (although at lower levels than in the brain), including skeletal muscle, heart, kidney, and testis [22]. To see whether accumulation of this PrPSc-like protein had pathologic consequences, we carried out histologic and functional analyses of peripheral tissues, and discovered that Tg(PG14) mice display a progressive, primary skeletal myopathy characterized by the presence of central nuclei, necrotic and regenerating myofibers, and variable fiber size [29]. Interestingly, cardiac tissue was histologically normal, as was cardiac function assayed by echocardiography. Because expression levels of PG14 PrP are similar in skeletal and cardiac muscle, these data suggest that cell types differ in their intrinsic susceptibility to pathologic effects of mutant PrP. Given the widespread distribution of protease-resistant and detergent-insoluble PrP throughout the central nervous system and periphery of Tg(PG14) mice, we concluded that structural features of the mutant protein itself, as opposed to cellular factors, play a primary role in its conversion to a PrPSc-like state.
Is PG14 PrP from Tg(PG14) mice infectious? The question of whether PG14 PrP from Tg(PG14) mice is infectious is a crucial one; an affirmative answer would prove that the mutant protein represents authentic PrPSc and would provide another demonstration of the protein-only theory of infectivity. Therefore, we inoculated brain homogenates from clinically ill Tg(PG14) mice of the A2 and A3 lines into several different hosts, including (1) low-expressing Tg(PG14-C) mice that do not become sick spontaneously, (2) Tg(WT) mice, and (3) non-Tg CD1 mice. None of the recipient mice became ill, and none displayed protease-resistant PrP in their brains by Western blotting during an observation period of 500 to 650 days postinoculation, at the end of which time significant numbers of animals were dying of intercurrent illness. Our conclusion from these data was that PG14 PrP produced in Tg mice either has a low titer of infectivity, or is not infectious at all (Chiesa R, Harris D, unpublished results, 2002).
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Interestingly, when Tg(PG14) mice were inoculated with a mouseadapted Rocky Mountain Laboratory strain of scrapie, they accumulated a form of PG14 PrP that was resistant to high concentrations of PK, and was infectious upon serial passage (Chiesa R, Harris D, unpublished results 2002). Thus, two forms of mutant PrP with identical primary sequences, but different degrees of protease resistance and infectivity are produced in Tg(PG14) mice, depending on whether the protein accumulates spontaneously or is ‘‘seeded’’ by exogenous prions. Structural analysis of these two isoforms will provide important insights into the molecular features of PrP that are essential for pathogenicity and the modifications associated with acquisition of infectivity. These studies highlight what has recently become a crucial question in the prion field—namely, whether PrPSc itself is the pathogenic form of PrP. Increasing evidence now suggests that although PrPSc is the infectious form of the protein, it is not the form that ultimately kills neurons [30]. We hypothesize that the weakly PK-resistant form of PrP that accumulates in Tg(PG14) mice is an example of a pathogenic but noninfectious form of PrP that may be present during the course of all prion diseases.
Summary We have produced a mouse model of a familial prion disorder by introduction of a transgene that encodes the moPrP homolog of a nineoctapeptide insertional mutant associated with an inherited form of CJD in humans. These mice develop progressive neurologic symptoms, display neuropathologic changes, and accumulate a form of mutant PrP in their brains and peripheral tissues that displays some of the biochemical properties of PrPSc. These mice have been extremely valuable for analyzing the cellular and biochemical mechanisms involved in inherited prion disorders and correlating the appearance of the PrPSc-like form with clinical and neuropathologic findings. Because the mutant protein in the mice is highly neurotoxic but appears to lack infectivity, further analysis of its properties promises to shed new light on the molecular distinction between pathogenic and infectious forms of PrP.
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