[The new research frontier on prion disease].
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Biomedical subjects
S Brandner
Publications and source records attributed to S Brandner.
[Significance of prion protein in transmission of prions and in pathogenesis of spongiform encephalopathies].
Prion disease or transmissible spongiform encephalopathies are caused by novel pathogens termed prions. Unlike classical infectious agents such as viruses or bacteria, prions lack an independent genome and consist largely if not entirely of an abnormal form of the host-encoded prion protein. How prions multiply is not known. A wealth of experimental evidence supports an essential role for the host-encoded prion protein in susceptibility and pathogenesis of prion diseases and in the propagation and spread of prions. In addition, B lymphocytes have been found to play a crucial role in the neuroinvasiveness of prions.
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Astrocyte-specific expression of hamster prion protein (PrP) renders PrP knockout mice susceptible to hamster scrapie.
Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte-specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 +/- 5 days and died 7 +/- 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.
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PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain.
Much available evidence points to a pathological isoform of the prion protein PrP being the infectious agent that causes transmissible spongiform encephalopathies, but the mechanisms controlling the neurotropism of prions are still unclear. We have previously shown that mice that do not express PrP (Prnp[o/o] mice) are resistant to infection by prions, and that if a Prnp(+/+) neurograft is introduced into such animals and these are infected intracerebrally with scrapie, the graft but not the surrounding tissue shows scrapie pathology. Here we show that PrP-expressing neurografts in Prnp(o/o) mice do not develop scrapie histopathology after intraperitoneal or intravenous inoculation with scrapie prions. Prion titres were undetectable in spleens of inoculated Prnp(o/o) mice, but were restored to wild-type levels upon reconstitution of the host lymphohaemopoietic system with PrP-expressing cells. Surprisingly, however, i.p. or i.v. inoculation failed to produce scrapie pathology in the neurografts of 27 out of 28 reconstituted animals, in contrast to intracerebral inoculation. We conclude that transfer of infectivity from the spleen to the central nervous system is crucially dependent on the expression of PrP in a tissue compartment that cannot be reconstituted by bone marrow transfer. Thus the requirement for the normal isoform of PrP in peripheral tissues represents a bottleneck for the spread of prions from peripheral sites to the central nervous system.
Bone Marrow↗
Tracking prions: the neurografting approach.
The physical nature of the agent that causes transmissible spongiform encephalopathies (the 'prion'), is the subject of passionate controversy. Investigation of it has benefited tremendously from the use of transgenic and knockout technologies. However, prion diseases present several other enigmas, including the mechanism of brain damage and how the affinity of the agent for the central nervous system is controlled. Here we show that such questions can be effectively addressed in transgenic and knockout systems, and that pathogenesis may be clarified even before we can be certain about the nature of the infectious agent. Availability of mice overexpressing the Prnp gene (which encodes the normal prion protein) and Prnp knockout mice allows for selective reconstitution experiments aimed at expressing PrP in specific portions of the brain or in selected populations of hemato- and lymphopoietic origin. We summarize how such studies can offer insights into how prions administered to peripheral sites can gain access to central nervous tissue, and into the molecular requirements for spongiform brain damage.
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Neurotoxicity and neuroinvasiveness of prions.
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[Molecular pathogenesis of spongiform encephalopathy].
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Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system.
Mice devoid of PrPC (Prnp%) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnp% brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnp% mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnp% mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.
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Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice.
Human cytomegalovirus (HCMV) can cause debilitating, sometimes fatal, opportunistic infections in congenitally infected infants and in immunodeficient individuals such as patients with the acquired immunodeficiency syndrome (AIDS). Molecular mechanisms that determine cell type specificity of HCMV infection and latency are poorly understood. We recently described a transgenic mouse model for analysis of HCMV major immediate-early (IE) promoter regulation and showed that sites of IE promoter activity during murine embryogenesis correlate with known target tissues of congenital HCMV infection in human fetuses (Koedood et al., 1995). Among various permissive human tissues, the brain is a site where HCMV infections can be devastating. Here, we have used immunohistochemical double-labeling analysis to identify specific cell types with HCMV-IE promoter activity in brains of transgenic mice at several postnatal stages. IE promoter activity was restricted to some endothelial cells, ependymal cells, choroid plexus epithelia, and neurons at discrete locations in the forebrain, brainstem, and cerebellum. Endothelial cells and neurons with activity were proportionately more abundant in neonatal than in adult brains. Although the IE promoter was normally silent in most astrocytes, activity was strongly induced in reactive astrocytes in response to a neocortical stab lesion. The findings support a model, consistent with clinical literature on HCMV encephalitis, whereby tissue damage and gliosis caused by HCMV infection of endothelial and ependymal cells progressively renders adjacent permissive neurons and reactive astrocytes accessible to infection. This transgenic model system should facilitate identification of factors that regulate the HCMV IE promoter with regard to infection permissivity and reactivation from latency.
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Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.
The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).
Amino Acid Sequence↗
Analysis of the determinants of neurotropism and neurotoxicity of HFV in transgenic mice.
The Bel-1 protein of human foamy virus (HFV) is a transactivator acting on the U3 region of the long terminal repeat and on an internal promoter (IP) immediately upstream of the bel genes. An HFV transgene called delta gpe, containing both promoters and all bel genes, is expressed in the central nervous system and induces neurodegeneration in mice. To dissect the role of individual promoters and bel genes on transgene expression and neurotoxicity we generated transgenic mice with a construct termed pL-bel1, which lacks the IP and the ancillary genes except bel-1. L-bel1 mice transcribed the HFV transgene in more tissues than delta gpe mice, suggesting that CNS specificity is dictated by cis-acting elements not present in the pLbel-1 construct. Unlike delta gpe mice, L-bel1 mice did not develop neurodegenerative changes and did not show induction of nitric oxide synthase expression, although both strains expressed Bel-1 in the brain. Therefore, Bel-1 expression is not sufficient for neurotoxicity. Our results suggest that Bet, a fusion protein between bel-1 and bel-2 which is highly expressed in delta gpe but not in L-bel1 mice, is a candidate for neurotoxicity.
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Normal host prion protein necessary for scrapie-induced neurotoxicity.
Accumulation of the prion protein PrPSc, a pathological and protease-resistant isoform of the normal host protein PrPC, is a feature of prion disease such as scrapie. It is still unknown whether scrapie pathology comes about by neurotoxicity of PrPSc, acute depletion of PrPC, or some other mechanism. Here we investigate this question by grafting neural tissue overexpressing PrPC into the brain of PrP-deficient mice which are scrapie-resistant and do not propagate infectivity. After intracerebral inoculation with scrapie prions, the grafts accumulated high levels of PrPSc and infectivity and developed the severe histopathological changes characteristic of scrapie. Moreover, substantial amounts of graft-derived PrPSc migrated into the host brain. Even 16 months after inoculation no pathological changes were seen in PrP-deficient tissue, not even in the immediate vicinity of the grafts. Therefore, in addition to being resistant to scrapie infection, brain tissue devoid of PrPC is not damaged by exogenous PrPSc.
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Mice homozygous for a modified beta-amyloid precursor protein (beta APP) gene show impaired behavior and high incidence of agenesis of the corpus callosum.
The amyloid precursor protein (beta APP) gene of the mouse was disrupted by homologous recombination; however, contrary to expectation, brain and other tissues still contained beta APP-specific RNA, albeit at a level 5-10 fold lower than wild-type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum.
Agenesis of Corpus Callosum↗
Microsurgical management of lateral lumbar disc herniations: combined lateral and interlaminar approach.
Seventy-three patients presenting either with biradicular symptoms caused by involvement of the upper and lower root or with monoradicular symptoms caused by affection of the upper root were treated between January 1993 and July 1995 in our department. An interlaminar and lateral access was used to decompress both the upper and lower root by combining the conventional interlaminar approach and a lateral partial facetectomy. With this technique, satisfactory to excellent results were obtained in 92% of the patients. The advantages of the combined approach are (i) optimized visualization of the disc and surrounding anatomical structures, (ii) improved exposure of the lateral foramen and thorough removal of disc material, (iii) minimal risk of root injury by improved visualization, (iv) preservation of a functional facet joint and thereby reduction of postoperative instability with persistent back pain. Since occasionally lateral disc herniations are poorly visualized by computed tomography or magnetic resonance imaging, the decision to use the combined approach should be guided by the patient's clinical presentation rather than by radiological findings.
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Transgenic and knockout mice in the study of neurodegenerative diseases.
Accurate animal models are essential for detailed analysis of the mechanisms underlying human neurodegenerative diseases. In addition, they can offer useful paradigms for the development and evaluation of new therapeutic strategies. We review the most popular techniques for modification of the mammalian genome in vivo, and provide a critical evaluation of the available transgenic mouse models for several neurological conditions of humans, including prion diseases, human retroviral diseases, Alzheimer's disease, and motor neuron diseases.
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Porphobilinogen deaminase deficiency in mice causes a neuropathy resembling that of human hepatic porphyria.
Acute intermittent porphyria (AIP) is a human disease resulting from a dominantly inherited partial deficiency of the heme biosynthetic enzyme, porphobilinogen deaminase (PBGD). The frequency of the trait for AIP is 1/10,000 in most populations, but may be markedly higher (1/500) in psychiatric patients. The clinical expression of the disease is characterized by acute, life-threatening attacks of 'porphyric neuropathy' that include abdominal pain, motor and sensory neurological deficits and psychiatric symptoms. Attacks are frequently precipitated by drugs, alcohol and low caloric intake. Identical symptoms occur in other hepatic porphyrias. To study the pathogenesis of the neurologic symptoms of AIP we have generated Pbgd-deficient mice by gene targeting. These mice exhibit the typical biochemical characteristics of human AIP, notably, decreased hepatic Pbgd activity, increased delta-aminolevulinic acid synthase activity and massively increased urinary excretion of the heme precursor, delta-aminolevulinic acid after treatment with drugs such as phenobarbital. Behavioural tests reveal decreased motor function and histopathological findings include axonal neuropathy and neurologic muscle atrophy.
Aminolevulinic Acid↗
Telencephalic transplants in mice: characterization of growth and differentiation patterns.
Telencephalic grafting represents a powerful tool for developmental studies and for the investigation of biological features of transgenic brain tissue. The interpretation of grafting experiments, however, requires detailed knowledge of graft biology. Therefore, we have characterized growth rates, graft size, and differentiation of embryonic telencephalic tissue harvested at various developmental stages and grafted into the caudoputamen and lateral ventricles of histocompatible mice. A total of 164 grafts were analysed up to 500 days after transplantation. Of all transplants, 79.3% resulted in the formation of solid neural grafts. Grafted cells were identified by 3H-thymidine labelling and autoradiography. Proliferation was studied by bromodeoxyuridine incorporation and decreased from an initial 35% at 1-3 d after grafting to less than 1.6% after 40 days. The graft size was measured as a function of the embryonic age of the transplanted tissue. Our data indicate that telencephalic tissue harvested at embryonic day E 12.5 reproducibly yields large, fully differentiated neuroectodermal grafts. The parameters defined in this study will be useful for detailed analysis of neuroectodermal tissue from mice undergoing fatal neurodegeneration, such as knockout mice bearing lethal mutations.
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Comparative in vivo and pathological analysis of the blood-brain barrier in mouse telencephalic transplants.
The post-transplantation status of the blood-brain barrier (BBB) is still a matter of debate. In an attempt to define BBB properties after neural transplantation in mice of a defined genetic background, we have used two exogenous markers (horseradish peroxidase and Evans blue), one endogenous marker (immunoglobulins), and in vivo contrast enhanced magnetic resonance imaging (MRI) and compared the results obtained with the different methods. With all four techniques employed, we found the BBB to be reconstituted in 67% of the grafts 3 weeks after grafting, and in more than 90% of all grafts 50 days after grafting. Horseradish peroxidase and contrast enhanced MRI were the most sensitive techniques, the latter offering the unique advantage of repetitive scanning of individual grafts. Our findings provide important information for transplantation studies in mouse models for neurodegenerative diseases.
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