Adriano Aguzzi

Mice devoid of PrP are resistant to scrapie

S.B. Prusiner proposed that the infectious agent of scraple, the prion, is PrPSc, a modified form of the normal host protein PrPC. Prn-p0/0 mice devoid of PrPC showed normal development and behavior. When inoculated with mouse scrapie prions, they remained free of scrapie symptoms for at least 13 months while wild-type controls all died within 6 months. Surprisingly, heterozygous Prn-p0/+ mice also showed enhanced resistance to scrapie. After introduction of Syrian hamster PrP transgenes, Prn-p0/0 mice became highly susceptible to hamster but not to mouse prions. These experiments show that PrPC, possibly at close to normal levels, is required for the usual susceptibility to scrapie and that lack of homology between incoming prions and the hosts PrP genes retards disease.

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).

Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.

Double‐stranded RNA‐dependent protein kinase (PKR) has been implicated in interferon (IFN) induction, antiviral response and tumor suppression. We have generated mice devoid of functional PKR (Pkr%). Although the mice are physically normal and the induction of type I IFN genes by poly(I).poly(C) (pIC) and virus is unimpaired, the antiviral response induced by IFN‐gamma and pIC was diminished. However, in embryo fibroblasts from Pkr knockout mice, the induction of type I IFN as well as the activation of NF‐kappa B by pIC, were strongly impaired but restored by priming with IFN. Thus, PKR is not directly essential for responses to pIC, and a pIC‐responsive system independent of PKR is induced by IFN. No evidence of the tumor suppressor activity of PKR was demonstrated.

An analytical solution to the kinetics of breakable filament assembly.

Dissecting Amyloid Formation Amyloid fibrils are associated with clinical disorders ranging from Alzheimers disease to type II diabetes. Their self-assembly can be described by a master equation that takes into account nucleation-dependent polymerization and fragmentation. Knowles et al. (p. 1533) now present an analytical solution to the master equation, which shows that amyloid growth kinetics is often limited by the fragmentation rate rather than by the rate of primary nucleation. In addition, the results reveal relationships between system properties (scaling laws) that provide mechanistic insight not only into amyloid growth, but also into related self-assembly processes. The growth kinetics of amyloid fibrils and related self-assembly phenomena are revealed by analytical theory. We present an analytical treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous molecular structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.

Experimental autoimmune encephalomyelitis repressed by microglial paralysis

Although microglial activation occurs in inflammatory, degenerative and neoplastic central nervous system (CNS) disorders, its role in pathogenesis is unclear. We studied this question by generating CD11b-HSVTK transgenic mice, which express herpes simplex thymidine kinase in macrophages and microglia. Ganciclovir treatment of organotypic brain slice cultures derived from CD11b-HSVTK mice abolished microglial release of nitrite, proinflammatory cytokines and chemokines. Systemic ganciclovir administration to CD11b-HSVTK mice elicited hematopoietic toxicity, which was prevented by transfer of wild-type bone marrow. In bone marrow chimeras, ganciclovir blocked microglial activation in the facial nucleus upon axotomy and repressed the development of experimental autoimmune encephalomyelitis. We conclude that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions. The microglial compartment thus provides a potential therapeutic target in inflammatory CNS disorders. These results validate CD11b-HSVTK mice as a tool to study the impact of microglial activation on CNS diseases in vivo.

Mammalian Prion Biology: One Century of Evolving Concepts

Prions have been responsible for an entire century of tragic episodes. Fifty years ago, kuru decimated the population of Papua New Guinea. Then, iatrogenic transmission of prions caused more than 250 cases of Creutzfeldt-Jakob disease. More recently, transmission of bovine spongiform encephalopathy to humans caused a widespread health scare. On the other hand, the biology of prions represents a fascinating and poorly understood phenomenon, which may account for more than just diseases and may represent a fundamental mechanism of crosstalk between proteins. The two decades since Stanley Prusiners formulation of the protein-only hypothesis have witnessed spectacular advances, and yet some of the most basic questions in prion science have remained unanswered.

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.

p62 Is a Common Component of Cytoplasmic Inclusions in Protein Aggregation Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimers, and Lewy bodies in Parkinsons disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.

Microglia: scapegoat, saboteur, or something else?

Microglia are resident immune cells in the brain and spinal cord. These cells provide immune surveillance and are mobilized in response to disparate diseases and injuries. Although microglial activation is often considered neurotoxic, microglia are essential defenders against many neurodegenerative diseases. It also seems increasingly likely that microglial dysfunction can underlie certain neurological diseases without an obvious immune component.

The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice.

High mobility group 1 (HMG1) protein is an abundant component of all mammalian nuclei, and related proteins exist in all eukaryotes. HMG1 binds linear DNA with moderate affinity and no sequence specificity, but bends the double helix significantly on binding through the minor groove. It binds with high affinity to DNA that is already sharply bent, such as linker DNA at the entry and exit of nucleosomes; thus, it is considered a structural protein of chromatin. HMG1 is also recruited to DNA by interactions with proteins required for basal and regulated transcription and V(D)J recombination. Here we generate mice harbouring deleted Hmg1. Hmg1–/– pups are born alive, but die within 24 hours due to hypoglycaemia. Hmg1-deficient mice survive for several days if given glucose parenterally, then waste away with pleiotropic defects (but no alteration in the immune repertoire). Cell lines lacking Hmg1 grow normally, but the activation of gene expression by the glucocorticoid receptor (GR, encoded by the gene Grl1) is impaired. Thus, Hmg1 is not essential for the overall organization of chromatin in the cell nucleus, but is critical for proper transcriptional control by specific transcription factors.