Nadia L. Tuzi

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann–Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild‐type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild‐type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild‐type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease‐associated PrP.

Host PrP Glycosylation: A Major Factor Determining the Outcome of Prion Infection

The expression of the prion protein (PrP) is essential for transmissible spongiform encephalopathy (TSE) or prion diseases to occur, but the underlying mechanism of infection remains unresolved. To address the hypothesis that glycosylation of host PrP is a major factor influencing TSE infection, we have inoculated gene-targeted transgenic mice that have restricted N-linked glycosylation of PrP with three TSE strains. We have uniquely demonstrated that mice expressing only unglycosylated PrP can sustain a TSE infection, despite altered cellular location of the host PrP. Moreover we have shown that brain material from mice infected with TSE that have only unglycosylated PrPSc is capable of transmitting infection to wild-type mice, demonstrating that glycosylation of PrP is not essential for establishing infection within a host or for transmitting TSE infectivity to a new host. We have further dissected the requirement of each glycosylation site and have shown that different TSE strains have dramatically different requirements for each of the glycosylation sites of host PrP, and moreover, we have shown that the host PrP has a major role in determining the glycosylation state of de novo generated PrPSc.

Expression of doppel in the CNS of mice does not modulate transmissible spongiform encephalopathy disease.

Late onset ataxia reported in three independently derived PrP null lines of mice has been attributed to the overexpression of the doppel protein in the CNS of these mice rather than to the loss of PrP. The central role of PrP in the transmissible spongiform encephalopathies (TSEs), the proximity of the gene which encodes doppel (Prnd) to the PrP gene (Prnp) and the structural similarity shared by PrP and doppel have led to the proposition that ataxia which develops during TSE disease could, in part, be due to doppel. In order to address this hypothesis, we have crossed our two inbred lines of PrP null mice, which either express (RCM) or do not express (NPU) the Prnd gene in the CNS, with mice expressing two Prnp(a[108F189V]) alleles of the PrP gene. We have found that the TSE infection does not influence the level of expression of Prnd in the CNS at the terminal stages of disease. Moreover, we have demonstrated that the level of expression of Prnd in the CNS has no influence on the incubation period, vacuolar pathology nor amount or distribution of PrP(Sc) deposition in the brains of the TSE-infected mice. Doppel has therefore no apparent influence on the outcome of TSE disease in transgenic mice, suggesting it is unlikely to be involved in the naturally occurring TSE diseases in other species.

Altered glycosylated PrP proteins can have different neuronal trafficking in brain but do not acquire scrapie-like properties

N-Linked glycans have been shown to have an important role in the cell biology of a variety of cell surface glycoproteins, including PrP protein. It has been suggested that glycosylation of PrP can influence the susceptibility to transmissible spongiform encephalopathy and determine the characteristics of the many different strains observed in this particular type of disease. To understand the role of carbohydrates in influencing the PrP maturation, stability, and cell biology, we have produced and analyzed gene-targeted murine models expressing differentially glycosylated PrP. Transgenic mice carrying the PrP substitution threonine for asparagine 180 (G1) or threonine for asparagine 196 (G2) or both mutations combined (G3), which eliminate the first, second, and both glycosylation sites, respectively, have been generated by double replacement gene targeting. An in vivo analysis of altered PrP has been carried out in transgenic mouse brains, and our data show that the lack of glycans does not influence PrP maturation and stability. The presence of one chain of sugar is sufficient for the trafficking to the cell membrane, whereas the unglycosylated PrP localization is mainly intracellular. However, this altered cellular localization of PrP does not lead to any overt phenotype in the G3 transgenic mice. Most importantly, we found that, in vivo, unglycosylated PrP does not acquire the characteristics of the aberrant pathogenic form (PrPSc), as was previously reported using in vitro models.

Glycosylation of PrPC Determines Timing of Neuroinvasion and Targeting in the Brain following Transmissible Spongiform Encephalopathy Infection by a Peripheral Route

ABSTRACT Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the periphery to the central nervous system where pathological lesions are formed. Several routes and cells within the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein cellular PrP (PrPC) is considered a key factor for replication of infectivity in the central nervous system (CNS) and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms of PrP have in these processes remain to be determined. To address this issue, we have used our unique models of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoculated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the glycosylation status of host PrP.

A single amino acid alteration in murine PrP dramatically alters TSE incubation time.

In order to investigate mutations linked to human TSEs, we have used the technique of gene targeting to introduce specific mutations into the endogenous murine PrP gene which resulted in a P101L substitution (Prnp(a101L)) in the murine PrP gene. This mutation is equivalent to the 102L mutation in the human PrP gene which is associated with Gerstmann-Sträussler syndrome. Since the mutated gene is in the correct chromosomal location and control of the mutant gene expression is identical to that of the wild type murine PrP gene, the precise effect of the 101L mutation in the uninfected and TSE infected mouse can be investigated in this transgenic model. Mice homozygous for this mutation (101LL) while showing no spontaneous TSE disease were more susceptible to TSE disease than wild type mice following inoculation with GSS infectivity. Disease was transmitted from these mice to mice both with and without the Prnp(a101L) allele. The 101L mutation does not therefore produce spontaneous genetic disease in mice but does dramatically alter incubation periods following TSE infection. Additionally, a rapid TSE transmission was demonstrated associated with extremely low amounts of PrP(Sc).

Dramatic Reduction of PrPC Level and Glycosylation in Peripheral Nerves following PrP Knock-Out from Schwann Cells Does Not Prevent Transmissible Spongiform Encephalopathy Neuroinvasion

Expression of the prion protein (PrPC) is a requirement for host susceptibility to the transmissible spongiform encephalopathies (TSEs) and thought to be necessary for the replication and transport of the infectious agent. The mechanism of TSE neuroinvasion is not fully understood, although the routing of infection has been mapped through the peripheral nervous system (PNS) and Schwann cells have been implicated as a potential conduit for transport of the TSE infectious agent. To address whether Schwann cells are a requirement for spread of the TSE agent from the site of infection to the CNS, PrPC expression was selectively removed from Schwann cells in vivo. This dramatically reduced total PrPC within peripheral nerves by 90%, resulting in the selective loss of glycosylated PrPC species. Despite this, 139A and ME7 mouse-passaged scrapie agent strains were efficiently replicated and transported to the CNS following oral and intraperitoneal exposure. Thus, the myelinating glial cells within the PNS do not appear to play a significant role in TSE neuroinvasion.

The role of host PrP in control of incubation time

PrP is central to TSE disease and has been hypothesised to be the infectious agent. Polymorphisms in the PrP gene are associated with different incubation times of disease following exposure to an infectious agent and mutations in the human PrP gene can apparently lead to spontaneous genetic disease. Strains of TSE agent are proposed to be generated and maintained through differences in glycosylation or conformation of PrP and the barrier to infection between species is thought to be due to the differences in the sequence of PrP between different species. To test these hypotheses, we have introduced specific modifications into the endogenous mouse Prnp gene by gene targeting. The mutated PrP gene is in the correct location under the control of the endogenous Prnp regulatory sequences and thus expressed in the same tissues and amounts as the wild type Prnp gene. By altering the murine PrP coding region to that of another species we have established that increasing overall identity between host and donor PrP can lead to either an increase or a decrease in incubation time of disease in a strain dependent manner. We have introduced a point mutation (101L) into the N-terminus of the host PrP and shown that it dramatically changes the susceptibility of the host to infection from different species. We have in addition demonstrated that polymorphisms in the N terminus (L108T) and C-terminus (F189V) of host PrP both alter the incubation time of disease. We have in addition introduced mutations into the Prnp gene which prevent glycosylation at each or both of the two N-linked glycosylation sites of PrP. Inoculation of these mice with infectivity has established that glycosylation of host PrP can influence incubation time of disease, vacuolar pathology and strain determination.