- Email: [email protected]
The puzzle of PrPSc and infectivity — do the pieces fit?
ett
rs
the
edito
neuron and applying the simulated conductance to the postsynaptic neuron, The dynamicclamp user interface provides straightforward conductance definition screens, in which the user specifies the voltage- and time-dependent parameters of the conductance(s) to be implemented. These parameters can be quickly and easily changed during an experiment. Additionally, our technique benefits from the use of readily available and widely used hardware - the Scientific Solutions Labmaster DMA system packaged in a TL-1 interface produced by Axon Instruments. High sampling rates might be useful in simulating fast conductances in neurons which can be
described by a single compartment. Since Robinson and Kawai, with their conductance 'pasting' technique, perform only a single linear real time calculation (driving force) they could realize a dramatic speed increase by taping their waveforms and replaying them through a simple analog circuit which would perform the driving force calculation and control the current injected into their neurons, thereby eliminating their need for a computer to control their method. While we have used such an analog circuit to simulate electrical synapses3, we cannot practically do so with many of the conductances implemented with the dynamic clamp, given their complex voltage and time dependencies. In
The puzzle of PrPsc and infectivity - do the pieces fit?
strains. Furthermore, the capacity arations of PrPsc contain 105 PrPsc to maintain distinct scrapie strains molecules per infectious unit 12, in mice and hamsters with the the proposal that each infectious same Prn-p allele suggests that unit contains only two to three the differences noted among PrPsc molecules suggests that scrapie strains passaged in mice virtually all PrPsc molecules are with different Pm-p alleles need non-infectious 12. There are two not necessarily be a function of concepts that evolve from this the PrP host-coded information, relationship between infectious The above discussion is not in- and non-infectious PrPsc moltended to discount the role of ecules. First, the change from differences in the PrP coding se- PrPc to PrPsc would not in itself quence in the outcome of agent- yield an infectious entity since the host interactions, but rather is majority of PrPsc molecules are intended to stress that information non-infectious. Second, if the incontained within the infectious fectious component of a partially agent affects the process in a purified PrPsc preparation is only fundamental way. a minute portion of the material On another point, the authors being examined, then the biostate that there is an '... estimate chemical, biophysical and conthat one infectious unit contains formational analyses of these 105 PrP molecules '9. This is used preparations might not yield into support the concept that the formation that is relevant to the capacity to form amyloid might infectious component. Furtherbe a key difference that dis- more, trying to assess the roles of tinguishes PrPc from PrPsc and infectious compared to nonthat the amyloidogenic nature of infectious PrPsc in pathological PrPsc might contribute to 'repli- changes, such as amyloid plaque cation, neuropathogenesis and formation, vacuolation and astrotransmissibility'. The estimate that cytosis, would be difficult - that 105 PrP molecules are contained is, do these pathological changes in each infectious unit 9 has been occur because of effects induced modified in recent communiby infectious PrPsc, non-infectious cations from the same group. PrPsc, or both? This issue is further Based upon partitioning of PrPsc clouded by the fact that recent molecules and infectivity in lipo- evidence (K. Hsiao, pers. comsomes, and on ionizing-radiation mun.) firmly establishes that mice target size, it is now thought that having the GSS mutant transeach infectious unit contains only gene develop neurodegenerative two to three PrPsc moleculeslO, ~1 changes, but do not contain Since partially purified prep- protease-resistant PrP (although
In a recent article 1 Ridley and Baker discuss the replication of prion proteins. We believe that several findings are pertinent to the authors' interpretations. Foremost among these issues is their comment that '... breeds of mice that typically sustain different "strains" of infectious agent have different Prn-p alleles'. The authors fail to note that there is extensive documentation that multiple scrapie strains can be 'sustained' in animal strains having the same Prn-p allele2-8; in fact, multiple scrapie strains can be maintained and phenotypic differences (for example, incubation period, histopathological changes and induction of obesity) can be observed in a single hamster strain 4'5'8 or in a specific inbred mouse strain 2,3,5-7. The authors use the fact that different strains of scrapie are maintained in mice having different Prn-p alleles, to buttress the concept that there is no need for a component of the infectious agent other than PrPsc. In contrast, the finding that distinct scrapie strains can be maintained and assessed in animals with the same Prn-p allele indicates that there is an additional component required to provide the information necessary to specify particular scrapie 48
. . . . . . . . . . . . general though, the reported sampling rate of the dynamic clamp is completely satisfactory, because the cable effects resulting from the structure of most neurons filters the higher frequencies from the recorded membrane potential. A. A, Sharp M. B. O'Neil L. F. Abbott Eve/Harder
Dept of Biology, BrandeisUniversity, Bassine 235, Waltham,/HA02254, USA.
References 1 Robinson, H. P. C. and Kawai, N. (1993) J. Neurosci. Methods 49, 157-165 2 Sharp, A. A., O'Neil, M, B., Abbott, L. F. and Marder, E. (1993) Trends Neurosci. 16, 389-394 3 Sharp, A. A. et aL (1992) J. Neurophysiol. 67, 1691-1694
TINS, VoL 17, No. 4, 1994
the original publication suggested PrPsc have not been found ~2. A that PrPsc was present) ~3, calling role for conformational differinto question the role this change ences is also problematical - it is (PrPc to PrPso) plays in patho- difficult to see how conformational differences could acgenesis. Our contention is that the commodate the number of nature of the agents causing un- scrapie strains and isolates that conventional slow infections is can be distinguished from each not established. In our view, the other following serial passages in key issue to be addressed is how a single P m - p genotype. For scrapie strains retain their distinc- example, in our own work we tive characteristics after multiple have demonstrated distinct difpassages in mice with the same ferences among three strains and Prn-p genotypes. These results five recent sheep isolates, all of indicate that there is a component which have been passaged at of the infectious agent that im- least three times in C57BL parts information that specifies mice 6'7'~4. The Edinburgh group, the characteristics of different using the same inbred mouse scrapie strains: for the virus hy- strain for serial passages, has pothesis6'7, this would be nucleic studied several additional strains acid with the capacity to code for that are distinguishable from the virus-specific proteins, and for the scrapie strains and isolates we virino hypothesis6'7, this would be have examined 2'3. a small, non-coding nucleic acid It is our firm view that the that uses host-specified protein nature of the agents causing un(perhaps PrP) as a protective coat, conventional slow infections and induces pathological changes remains one of the greatest by affecting regulatory mechan- puzzles in biology ~'~5. isms. The proponents of the prion hypothesis believe that differRichard L Carp ences in the PrP molecule (either Richard J. Kascsak post-translational modifications Richard Rubenstein or conformational differences) are Dept of Virology, NYS Institute for Basic capable of inducing the characResearch in Developmental Disabilities, 1050 teristics that distinguish scrapie ForestH#/ Road, StatenIsland, NY 19314, USA. strains and of maintaining these characteristics during serial Patricia A. Merz passages. They currently favor Dept of PathologicalNeurobto/ogy,NYSInstitute the conformational concept since for BasicResearchtn DevelopmentalDisab#ittes, post-translational modification 1050 ForestHi//Road, StatenIsland, NY 19314, differences between PrPc and USA.
References
Neu rotransmission - the link integrating Alzheimer research?
side effects compared with those receiving other medication (mostly antipsychotics). We have also reported that dementia, considered to be caused by hypoactivity of glutamatergic pyramidal neurones, is associated with reduced secretion and intracellular accumulation of APP, a proposal based on two observations. First, secreted APP is lower in the lumbar cerebrospinal fluid of demented patients with cortical pyramidal neurone loss compared with controls. This finding was independent of presynaptic cholinergic dysfunction and the presence of either senile plaques or neurofibrillary pathology. A plausible explanation for the presence of senile plaques in Alzheimer's
It is widely recognized that neurotransmitter replacement therapy is needed to ameliorate the cognitive impairment characteristic of AIzheimer's disease. Several groups, including our own ~'2, have provided evidence to support the idea outlined by Selkoe 3 that this approach will also beneficially affect the metabolism of the amyloid precursor protein (APP) by favouring the non-amyloidogenic pathway. Phosphorylation, stimulated by depolarization or second-messenger systems, might be enhanced by antagonists at the inhibitory TINS, Vol. 17, No. 4, 1994
5-HT1A receptor, or by activation of the phospholipase-C-linked cholinergic M1 receptor, both of which have been found to be enriched on cortical pyramidal neurones2. Concepts arising from this work on cultured cells 1,3 and animals modelling an aspect of Alzheimer's disease (selective cortical pyramidal neurone loss2) have recently been extended to humans. Here, negative regulation of phospholipase-C activity was found to be associated with lower concentrations of secreted non-amyloidogenic APP. This was based on ventricular cerebrospinal fluid samples obtained during air encephalography from patients receiving lithium salts and drugs with anti-cholinergic
1 Ridley,R. M. and Baker, H. F. (1993) Trends Neurosci. 16, 425-426 2 Dickinson, A. G., Bruce, M. E., Outram, G. W. and Kimberlin, R. H. (1984) in Proceedings of Workshop on
3
4 5 6
Slow
Transmissible Diseases
(Tateishi, J., ed.), pp. 105-118, Japanese Ministry of Health and Welfare Dickinson,A. G. and Fraser,H. (1979) in Slow Transmissible Diseases of the Nervous System, Vol. 1 (Prusiner,S. B. and Hadlow, W. J., eds), pp. 367-385, Academic Press De Armond, S. J. et aL (1993) Proc. Natl Acad. ScL USA 90, 6449-6453 Kimberlin, R. H., Walker, C. A. and Fraser, H. (1989)J. Gen. Virol. 70, 2017-2025 Kascsak, R. J., Rubenstein, R. and Carp, R. I. (1991) in Current Topics in Microbiology
and
Immunology
(Chesebro, B. W., ed.), pp. 139-152, Springer-Verlag 7 Carp, R. I. and Rubenstein,R. (1991) in Seminars in Virology (Haase,A. T., ed.), pp. 203-213, Academic Press 8 Carp, R. I., Kim, Y. S. and Callahan, S. M. (1990) J. Infect. Dis. 161, 462-466 9 Prusiner, S. B. et aL (1983) Cell 35, 349-358 10 Gabizon, R., McKinley, M. P. and Prusiner,S. B. (1987) Proc. Natl Acad. Sci. USA 84, 4017-4021 11 Bellinger-Kawahara,C. G., Kempner, E., Growth, D., Gabizon, R. and Prusiner, S. B. (1988) Virology 164, 533-541 12 Prusiner, S. B. (1991) Science 252, 1515-1522 13 Hsiao,K. K. etal. (1990) Science 250, 1587- 1590 14 Carp, R. I. and Callahan,S. M. (1991) J. Gen. Virol. 72, 293-298 15 Carp, R. I. et aL (1989) Alzhetmer's Dis. Assoc. Disord. 3, 79-99
149
rs
the
edito
neuron and applying the simulated conductance to the postsynaptic neuron, The dynamicclamp user interface provides straightforward conductance definition screens, in which the user specifies the voltage- and time-dependent parameters of the conductance(s) to be implemented. These parameters can be quickly and easily changed during an experiment. Additionally, our technique benefits from the use of readily available and widely used hardware - the Scientific Solutions Labmaster DMA system packaged in a TL-1 interface produced by Axon Instruments. High sampling rates might be useful in simulating fast conductances in neurons which can be
described by a single compartment. Since Robinson and Kawai, with their conductance 'pasting' technique, perform only a single linear real time calculation (driving force) they could realize a dramatic speed increase by taping their waveforms and replaying them through a simple analog circuit which would perform the driving force calculation and control the current injected into their neurons, thereby eliminating their need for a computer to control their method. While we have used such an analog circuit to simulate electrical synapses3, we cannot practically do so with many of the conductances implemented with the dynamic clamp, given their complex voltage and time dependencies. In
The puzzle of PrPsc and infectivity - do the pieces fit?
strains. Furthermore, the capacity arations of PrPsc contain 105 PrPsc to maintain distinct scrapie strains molecules per infectious unit 12, in mice and hamsters with the the proposal that each infectious same Prn-p allele suggests that unit contains only two to three the differences noted among PrPsc molecules suggests that scrapie strains passaged in mice virtually all PrPsc molecules are with different Pm-p alleles need non-infectious 12. There are two not necessarily be a function of concepts that evolve from this the PrP host-coded information, relationship between infectious The above discussion is not in- and non-infectious PrPsc moltended to discount the role of ecules. First, the change from differences in the PrP coding se- PrPc to PrPsc would not in itself quence in the outcome of agent- yield an infectious entity since the host interactions, but rather is majority of PrPsc molecules are intended to stress that information non-infectious. Second, if the incontained within the infectious fectious component of a partially agent affects the process in a purified PrPsc preparation is only fundamental way. a minute portion of the material On another point, the authors being examined, then the biostate that there is an '... estimate chemical, biophysical and conthat one infectious unit contains formational analyses of these 105 PrP molecules '9. This is used preparations might not yield into support the concept that the formation that is relevant to the capacity to form amyloid might infectious component. Furtherbe a key difference that dis- more, trying to assess the roles of tinguishes PrPc from PrPsc and infectious compared to nonthat the amyloidogenic nature of infectious PrPsc in pathological PrPsc might contribute to 'repli- changes, such as amyloid plaque cation, neuropathogenesis and formation, vacuolation and astrotransmissibility'. The estimate that cytosis, would be difficult - that 105 PrP molecules are contained is, do these pathological changes in each infectious unit 9 has been occur because of effects induced modified in recent communiby infectious PrPsc, non-infectious cations from the same group. PrPsc, or both? This issue is further Based upon partitioning of PrPsc clouded by the fact that recent molecules and infectivity in lipo- evidence (K. Hsiao, pers. comsomes, and on ionizing-radiation mun.) firmly establishes that mice target size, it is now thought that having the GSS mutant transeach infectious unit contains only gene develop neurodegenerative two to three PrPsc moleculeslO, ~1 changes, but do not contain Since partially purified prep- protease-resistant PrP (although
In a recent article 1 Ridley and Baker discuss the replication of prion proteins. We believe that several findings are pertinent to the authors' interpretations. Foremost among these issues is their comment that '... breeds of mice that typically sustain different "strains" of infectious agent have different Prn-p alleles'. The authors fail to note that there is extensive documentation that multiple scrapie strains can be 'sustained' in animal strains having the same Prn-p allele2-8; in fact, multiple scrapie strains can be maintained and phenotypic differences (for example, incubation period, histopathological changes and induction of obesity) can be observed in a single hamster strain 4'5'8 or in a specific inbred mouse strain 2,3,5-7. The authors use the fact that different strains of scrapie are maintained in mice having different Prn-p alleles, to buttress the concept that there is no need for a component of the infectious agent other than PrPsc. In contrast, the finding that distinct scrapie strains can be maintained and assessed in animals with the same Prn-p allele indicates that there is an additional component required to provide the information necessary to specify particular scrapie 48
. . . . . . . . . . . . general though, the reported sampling rate of the dynamic clamp is completely satisfactory, because the cable effects resulting from the structure of most neurons filters the higher frequencies from the recorded membrane potential. A. A, Sharp M. B. O'Neil L. F. Abbott Eve/Harder
Dept of Biology, BrandeisUniversity, Bassine 235, Waltham,/HA02254, USA.
References 1 Robinson, H. P. C. and Kawai, N. (1993) J. Neurosci. Methods 49, 157-165 2 Sharp, A. A., O'Neil, M, B., Abbott, L. F. and Marder, E. (1993) Trends Neurosci. 16, 389-394 3 Sharp, A. A. et aL (1992) J. Neurophysiol. 67, 1691-1694
TINS, VoL 17, No. 4, 1994
the original publication suggested PrPsc have not been found ~2. A that PrPsc was present) ~3, calling role for conformational differinto question the role this change ences is also problematical - it is (PrPc to PrPso) plays in patho- difficult to see how conformational differences could acgenesis. Our contention is that the commodate the number of nature of the agents causing un- scrapie strains and isolates that conventional slow infections is can be distinguished from each not established. In our view, the other following serial passages in key issue to be addressed is how a single P m - p genotype. For scrapie strains retain their distinc- example, in our own work we tive characteristics after multiple have demonstrated distinct difpassages in mice with the same ferences among three strains and Prn-p genotypes. These results five recent sheep isolates, all of indicate that there is a component which have been passaged at of the infectious agent that im- least three times in C57BL parts information that specifies mice 6'7'~4. The Edinburgh group, the characteristics of different using the same inbred mouse scrapie strains: for the virus hy- strain for serial passages, has pothesis6'7, this would be nucleic studied several additional strains acid with the capacity to code for that are distinguishable from the virus-specific proteins, and for the scrapie strains and isolates we virino hypothesis6'7, this would be have examined 2'3. a small, non-coding nucleic acid It is our firm view that the that uses host-specified protein nature of the agents causing un(perhaps PrP) as a protective coat, conventional slow infections and induces pathological changes remains one of the greatest by affecting regulatory mechan- puzzles in biology ~'~5. isms. The proponents of the prion hypothesis believe that differRichard L Carp ences in the PrP molecule (either Richard J. Kascsak post-translational modifications Richard Rubenstein or conformational differences) are Dept of Virology, NYS Institute for Basic capable of inducing the characResearch in Developmental Disabilities, 1050 teristics that distinguish scrapie ForestH#/ Road, StatenIsland, NY 19314, USA. strains and of maintaining these characteristics during serial Patricia A. Merz passages. They currently favor Dept of PathologicalNeurobto/ogy,NYSInstitute the conformational concept since for BasicResearchtn DevelopmentalDisab#ittes, post-translational modification 1050 ForestHi//Road, StatenIsland, NY 19314, differences between PrPc and USA.
References
Neu rotransmission - the link integrating Alzheimer research?
side effects compared with those receiving other medication (mostly antipsychotics). We have also reported that dementia, considered to be caused by hypoactivity of glutamatergic pyramidal neurones, is associated with reduced secretion and intracellular accumulation of APP, a proposal based on two observations. First, secreted APP is lower in the lumbar cerebrospinal fluid of demented patients with cortical pyramidal neurone loss compared with controls. This finding was independent of presynaptic cholinergic dysfunction and the presence of either senile plaques or neurofibrillary pathology. A plausible explanation for the presence of senile plaques in Alzheimer's
It is widely recognized that neurotransmitter replacement therapy is needed to ameliorate the cognitive impairment characteristic of AIzheimer's disease. Several groups, including our own ~'2, have provided evidence to support the idea outlined by Selkoe 3 that this approach will also beneficially affect the metabolism of the amyloid precursor protein (APP) by favouring the non-amyloidogenic pathway. Phosphorylation, stimulated by depolarization or second-messenger systems, might be enhanced by antagonists at the inhibitory TINS, Vol. 17, No. 4, 1994
5-HT1A receptor, or by activation of the phospholipase-C-linked cholinergic M1 receptor, both of which have been found to be enriched on cortical pyramidal neurones2. Concepts arising from this work on cultured cells 1,3 and animals modelling an aspect of Alzheimer's disease (selective cortical pyramidal neurone loss2) have recently been extended to humans. Here, negative regulation of phospholipase-C activity was found to be associated with lower concentrations of secreted non-amyloidogenic APP. This was based on ventricular cerebrospinal fluid samples obtained during air encephalography from patients receiving lithium salts and drugs with anti-cholinergic
1 Ridley,R. M. and Baker, H. F. (1993) Trends Neurosci. 16, 425-426 2 Dickinson, A. G., Bruce, M. E., Outram, G. W. and Kimberlin, R. H. (1984) in Proceedings of Workshop on
3
4 5 6
Slow
Transmissible Diseases
(Tateishi, J., ed.), pp. 105-118, Japanese Ministry of Health and Welfare Dickinson,A. G. and Fraser,H. (1979) in Slow Transmissible Diseases of the Nervous System, Vol. 1 (Prusiner,S. B. and Hadlow, W. J., eds), pp. 367-385, Academic Press De Armond, S. J. et aL (1993) Proc. Natl Acad. ScL USA 90, 6449-6453 Kimberlin, R. H., Walker, C. A. and Fraser, H. (1989)J. Gen. Virol. 70, 2017-2025 Kascsak, R. J., Rubenstein, R. and Carp, R. I. (1991) in Current Topics in Microbiology
and
Immunology
(Chesebro, B. W., ed.), pp. 139-152, Springer-Verlag 7 Carp, R. I. and Rubenstein,R. (1991) in Seminars in Virology (Haase,A. T., ed.), pp. 203-213, Academic Press 8 Carp, R. I., Kim, Y. S. and Callahan, S. M. (1990) J. Infect. Dis. 161, 462-466 9 Prusiner, S. B. et aL (1983) Cell 35, 349-358 10 Gabizon, R., McKinley, M. P. and Prusiner,S. B. (1987) Proc. Natl Acad. Sci. USA 84, 4017-4021 11 Bellinger-Kawahara,C. G., Kempner, E., Growth, D., Gabizon, R. and Prusiner, S. B. (1988) Virology 164, 533-541 12 Prusiner, S. B. (1991) Science 252, 1515-1522 13 Hsiao,K. K. etal. (1990) Science 250, 1587- 1590 14 Carp, R. I. and Callahan,S. M. (1991) J. Gen. Virol. 72, 293-298 15 Carp, R. I. et aL (1989) Alzhetmer's Dis. Assoc. Disord. 3, 79-99
149