Shrinking prions: new folds to old questions.
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Animals↗
Biomedical subjects
A Aguzzi
Publications and source records attributed to A Aguzzi.
Spinal cord pathology and viral burden in homosexuals and drug users with AIDS.
Unless treated with effective antiretroviral therapy many AIDS patients develop a characteristic vacuolar myelopathy of the spinal cord associated with moderate clinical disability. Opinion is divided as to whether vacuolar myelopathy is causally linked to HIV myelitis. To investigate this further, spinal cord pathology was assessed in 41 drug users, 33 homosexual men and 16 other patients, all with AIDS. Previous work has shown that HIV encephalitis is more common in Edinburgh drug users than in homosexual men. In the present study HIV myelitis (10% overall) was more common in drug users (17%) than in homosexual men (3%) (P = 0.05), whereas the incidence of opportunistic infections (7% v. 9%) and lymphomas (2% v. 6%) was comparable in the two groups, but with a slight trend in the reverse direction, reflecting similar findings in the brain. However, moderate or severe vacuolar myelopathy was equally represented in both groups (20% of drug users and 21% of homosexual men). The HIV proviral load, assessed by polymerase chain reaction in frozen samples of thoracic spinal cord in 37 cases, correlated closely with the presence of giant cells and/or with immunocytochemical evidence of productive HIV infection. In 13 cases, the proviral load was measured in cervical, thoracic and lumbar samples and proved to be uniformly high or low in individual cases. This study provides no evidence for direct involvement of HIV, cytomegalovirus, papovavirus or human foamy virus in the pathogenesis of vacuolar myelopathy.
AIDS-Related Opportunistic Infections↗
Distinct phases of cryogenic tissue damage in the cerebral cortex of wild-type and c-fos deficient mice.
To characterize the development of tissue damage following cryogenic injury to the mouse cortex, the time course of histopathological changes, transcriptional responses and DNA strand breaks following application of a liquid nitrogen-cooled probe to the surface of the parietal bone were assessed. Distinct phases of tissue damage were observed: after 30 min, there was demarcation of a core lesion followed by mainly necrotic cell death starting 2 h after injury. At 12 hours, progressive apoptotic death of scattered cells in the periphery of the core lesion was detected, resembling the penumbra observed in ischaemic stroke. In situ hybridization for c-fos revealed an absence of expression in the core region, suggesting early cessation of transcription. There was strong induction of c-fos in the penumbra 30 min after the lesion, which had spread over the ipsilateral hemisphere at 2 h, possibly caused by peri-infarction depolarization. At later time points, sustained expression of c-fos was observed in some cells in the penumbra. Since a role for c-fos has been postulated in the initiation or execution of apoptotic pathways, the susceptibility of c-fos deficient mice was explored (n=4) in this model. Cryoinjury-induced tissue injury was markedly attenuated in c-fos deficient mice. A model of the phases and mechanisms of cryogenic injury is proposed, which discriminates an early phase characterized by physical changes caused by hypothermia and their immediate consequences (i.e. transcriptional block), an intermediate phase where secondary changes lead to necrosis in the core region, and a final phase of delayed apoptotic cell death in the penumbra.
Animals↗
Distal axonopathy in peripheral nerves of PMP22-mutant mice.
A partial duplication of chromosome 17 is associated with Charcot-Marie-Tooth disease type 1A (CMT1A), a demyelinating peripheral neuropathy that causes progressive distal muscle atrophy and sensory impairment. Trisomic expression of peripheral myelin protein 22 (PMP22) whose gene is contained within the duplicated region is considered to be responsible for the disease. By using recombinant gene technology in rodents, we had demonstrated previously that PMP22 is sensitive to gene dosage. Homozygous PMP22 knockout (PMP22(0/0)) mice and transgenic animals carrying additional copies of the PMP22 gene develop distinct peripheral polyneuropathies. We have now performed a detailed morphometrical analysis of the L3 roots, quadriceps and saphenous nerves of these PMP22-mutant mice to study whether the myelin and potential axonal deficits are evenly distributed. The L3 roots and the peripheral nerves were chosen as representatives of the proximal and distal segments of the peripheral nervous system. When the roots were compared with the peripheral nerves, myelin deficiencies appeared more severe at the radicular levels, in particular the ventral roots. Decreased numbers of large calibre axons were a prominent feature in the motor branches of both strains of PMP22-mutant mice, and these axonal deficits were more severe distally. Active axonal damage was only observed in the nerves of PMP22(0/0) mice. Despite the distinct effects on myelination and the Schwann cell phenotype that characterize the neuropathies of PMP22-mutant mice, both strains develop a distally accentuated axonopathy as a common disease mechanism which is likely to be responsible for the neurological deficits.
Animals↗
No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.
Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.
Animals↗
Scrapie pathogenesis in subclinically infected B-cell-deficient mice.
Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.
Animals↗
Human MxA protein protects mice lacking a functional alpha/beta interferon system against La crosse virus and other lethal viral infections.
The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.
Alphavirus Infections↗
Biphasic edema after hypoxic-ischemic brain injury in neonatal rats reflects early neuronal and late glial damage.
Magnetic resonance imaging with diffusion- and T2-weighted imaging and 31P magnetic resonance spectroscopy was used to investigate the relationship between development of brain edema and alterations of the brain energy metabolism after hypoxia-ischemia (HI) brain injury in 7-d-old rats. The results were correlated with histologic examinations at various times during recovery up to 5 d. Moderate HI, induced by right common carotid artery ligation and subsequent exposure to 8% O2 for 90 min, produced a cytotoxic edema of 52+/-9% brain volume and depressed the ratio of phosphocreatine to inorganic phosphate from 1.43+/-0.21 to 0.11+/-0.09. Within 1 h of reoxygenation, the edema decreased to 4+/-2% of brain volume, demarcating the core of the lesion. At 5 h of recovery, a secondary cytotoxic edema together with a newly developing vasogenic edema expanded again, reaching its maximal extent of 45+/-10% brain volume at around 24 h. The ratio of phosphocreatine to inorganic phosphate recovered slowly, reaching 1.12+/-0.27 around 13 h. Thereafter it declined again in a manner analogous to the observations made in human newborns after severe perinatal asphyxia, reaching trough values of 0.48+/-0.22 around 24 h after HI. At the cellular level, the vast majority of neuronal death occurred before 15 h. Subsequently, strong glial activation lasted 2-3 d after HI. At 5 d, a cystic infarction of 35+/-12% brain volume was found. We conclude that the biphasic evolution of brain edema and energy metabolism reflects early neuronal and late glial damage in response to moderate HI injury. Therefore, the secondary energy breakdown reflects glial activation and subsequent glial death.
Animals↗
The heavy metal-responsive transcription factor-1 (MTF-1) is not required for neural differentiation.
The zinc finger transcription factor MTF-1 is essential for proper response to heavy metal load and other stress conditions in vertebrates, and also contributes to the maintenance of the cellular redox state. Target genes include metallothioneins (MT-I and MT-II) and gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme involved in glutathione biosynthesis. Although MTF-1 is expressed ubiquitously, the primary defect in null mutant mice is hepatocyte necrosis, which results in embryonic lethality around day E14 and prevents the analysis of delayed effects on other organs. To assess the impact of MTF-1 deficiency on the function of the mature central nervous system, we employed the neural grafting strategy. Neuroectodermal brain tissue obtained from transgenic mouse embryos at gestational day 12.5 was transplanted into the caudoputamen of adult wild-type mice. 33 days later, grafts derived from MTF-1 deficient mice consisted of fully differentiated neuroectodermal tissue and showed no differences to heterozygous control grafts. This indicates that MTF-1 is dispensable for the development and differentiation of the nervous system. Such transplants devoid of MTF-1 may provide a useful tool for the further investigation of the effect of cell stress, including oxidative stress.
Animals↗
Characterization of Tu-2449, a glioma cell line derived from a spontaneous tumor in GFAP-v-src-transgenic mice: comparison with established murine glioma cell lines.
Glial fibrillary acidic protein (GFAP)-v-src transgenic mice develop spontaneous gliomas with a high incidence of malignant progression. We characterize the first glial cell line derived from v-src transgenic mice, Tu-2449 in comparison with a virally induced murine glioma, SRB-10, and a spontaneous murine glioma, P497. Doubling times were lowest, as clonogenicity in soft agar was highest for Tu-2449, and to a lesser degree, Tu-2449 cells formed spheroids and showed migratory behaviour and invaded fetal rat brain aggregates. BCL-2 and BAX expression were lower in Tu-2449 and P497 than in SRB-10 cells. Only Tu-2449 cells accumulated p53 protein in response to genotoxic stress. Tu-2449 and SRB-10 cells that showed low CD95 expression were resistant to CD95 ligand (CD95L)-induced apoptosis unless coexposed to CD95L and inhibitors of RNA or protein synthesis. A chemosensitivity profile revealed Tu-2449 to be rather chemoresistant. Tu-2449 thus displays growth characteristics and patterns of resistance to apoptosis similar to those of other mouse and human glioma cell lines and may therefore become a valuable tool to evaluate new therapies for malignant gliomas in vitro and in vivo.
Animals↗
Grafting mouse brains: from neurocarcinogenesis to neurodegeneration.
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Animals↗
Prion protein expression in different species: analysis with a panel of new mAbs.
By immunizing prion knockout mice (Prnp-/-) with recombinant murine prion protein (PrPc), we obtained a panel of mAbs specific for murine PrPc. These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrPc) and protease-resistant (PrPres) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrPc. These newly generated PrPc antibodies will help to explore the biology of PrPc and to establish the diagnosis of prion diseases in both humans and animals.
Animals↗
A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain.
Measles viruses (MV) can be isolated from the brains of deceased subacute sclerosing panencephalitis patients only in a cell-associated form. These viruses are often defective in the matrix (M) protein and always seem to have an altered fusion protein cytoplasmic tail. We reconstituted a cell-free, infectious M-less MV (MV-DeltaM) from cDNA. In comparison with standard MV, MV-DeltaM was considerably more efficient at inducing cell-to-cell fusion but virus titres were reduced approximately 250-fold. In MV-DeltaM-induced syncytia the ribonucleocapsids and glycoproteins largely lost co-localization, confirming the role of M protein as the virus assembly organizer. Genetically modified mice were inoculated with MV-DeltaM or with another highly fusogenic virus bearing glycoproteins with shortened cytoplasmic tails (MV-Delta(tails)). MV-DeltaM and MV-Delta(tails) lost acute pathogenicity but penetrated more deeply into the brain parenchyma than standard MV. We suggest that enhanced cell fusion may also favour the propagation of mutated, assembly-defective MV in human brains.
Amino Acid Sequence↗
Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.
We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA).
Animals↗
Spongiform encephalopathies. The prion's perplexing persistence.
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Animals↗
Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar lesions.
The physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32-121 or 32-134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.
Alleles↗
A strain-independent postnatal neurodegeneration in mice lacking the EGF receptor.
Mice lacking the epidermal growth factor receptor (EGFR) exhibit strain-dependent phenotypes ranging from placental to postnatal skin, lung and brain defects. After birth, all mutant mice develop a progressive neurodegeneration in the frontal cortex, olfactory bulb and thalamus, characterized by massive apoptosis and upregulation of c-fos. These defects occur in a strain-independent manner, since neither rescue of the placental phenotype by aggregation of diploid 129/Sv EGFR mutant and tetraploid wild-type embryos, nor promotion of lung maturation by transplacental dexamethasone administration alters the course of neurodegeneration. VEGF is not induced during the degenerative process, excluding hypoxia and ischemia as causes of cell death. A migratory disorder is detected in the hippocampus with nests of ectopic neurons, which are also apoptotic. Cerebral cortices from EGFR mutants contain lower numbers of GFAP positive astrocytes, which display reduced proliferation in vitro. Since EGFR is expressed in the affected cell-types, these results define a specific function for EGFR in the proliferation and/or differentiation of astrocytes and in the survival of postmitotic neurons.
Animals↗
Protein conformation dictates prion strain.
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Immunoassay↗