Eckhard Flechsig

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.

Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody

Exposure of susceptible neuroblastoma N2a cells to mouse scrapie prions leads to infection, as evidenced by the continued presence of the scrapie form of the prion protein (PrPSc) and infectivity after 300 or more cell doublings. We find that exposure to phosphatidylinositol-specific phospholipase C (PIPLC) or to the monoclonal anti-prion protein (PrP) antibody 6H4 not only prevents infection of susceptible N2a cells but also cures chronically scrapie-infected cultures, as judged by the long-term abrogation of PrPSc accumulation after cessation of treatment. A nonpassaged, stationary infected culture rapidly loses PrPSc when exposed to the antibody or PIPLC, indicating that the PrPSc level is determined by steady state equilibrium between formation and degradation, and that depletion of the cellular form of PrP can interrupt the propagation of PrPSc. These findings encourage the belief that passive immunization may provide a therapeutic approach to prion disease.

A crucial role for B cells in neuroinvasive scrapie

Although prion proteins are most efficiently propagated through intracerebral inoculation, peripheral administration has caused the diseases kuru, iatrogenic Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and new-variant CJD,. The development of neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system,. Here we investigate the identity of these cells by using a panel of immune-deficient mice inoculated with prions intraperitoneally: we found that defects affecting only T lymphocytes had no apparent effect, but that all mutations that disrupted the differentiation and response of B lymphocytes prevented the development of clinical scrapie. As an absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells, a lack of any of these three components may prevent the development of clinical scrapie. However, we found that scrapie developed after peripheral inoculation in mice expressing immunoglobulins that were exclusively of the M subclass and without detectable specificity for the normal form of the prion PrPc, and in mice which had differentiated B cells but no functional follicular dendritic cells. We conclude that differentiated B cells are crucial for neuroinvasion by scrapie, regardless of the specificity of their receptors.

A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions.

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment. In a log/log plot, the dose–response is linear over two logs of prion concentrations. The SC assay is about as sensitive as the mouse bioassay, 10 times faster, >2 orders of magnitude less expensive, and suitable for robotization. SC assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

PrP expression in B lymphocytes is not required for prion neuroinvasion

Prion diseases are typically initiated by infection of peripheral sites, as in the case of bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, kuru and most cases of iatrogenic Creutzfeldt-Jakob disease. In mouse scrapie, prion infectivity accumulates in lymphoid organs, and the absence of mature B lymphocytes prevents peripherally administered prions from inducing central nervous system disease. We have now assessed whether expression of the cellular prion protein, PrPC, is required for B lymphocytes to mediate neuroinvasion. We found that repopulation of SCID and Rag-1-/- mice with fetal liver cells from either PrP-expressing or PrP-deficient mice and from T-cell deficient mice, but not from B-cell deficient mice, is equally efficient in restoring neuroinvasion after intraperitoneal inoculation of scrapie prions. These results indicate that cells whose maturation depends on B cells or their products, such as follicular dendritic cells, may enhance neuroinvasion. Alternatively, B cells may transport prions to the nervous system by a PrP-independent mechanism.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted (‘Zürich I’) remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, Prnd. A new PrP knockout line, ‘Zürich II’, with a 2.9 kb Prnp deletion, developed this phenotype at ∼10 months (50% morbidity). A single Prnp allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a Prnd‐containing cosmid expressed Dpl at twice the level and became ataxic ∼5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Prion Protein Devoid of the Octapeptide Repeat Region Restores Susceptibility to Scrapie in PrP Knockout Mice

Mice devoid of PrP are resistant to scrapie and fail to replicate the agent. Introduction of transgenes expressing PrP into such mice restores susceptibility to scrapie. We find that truncated PrP devoid of the five copper binding octarepeats still sustains scrapie infection; however, incubation times are longer and prion titers and protease-resistant PrP are about 30-fold lower than in wild-type mice. Surprisingly, brains of terminally ill animals show no histopathology typical for scrapie. However, in the spinal cord, infectivity, gliosis, and motor neuron loss are as in scrapie-infected wild-type controls. Thus, while the region comprising the octarepeats is not essential for mediating pathogenesis and prion replication, it modulates the extent of these events and of disease presentation.

PrP-dependent association of prions with splenic but not circulating lymphocytes of scrapie-infected mice.

An intact immune system, and particularly the presence of mature B lymphocytes, is crucial for mouse scrapie pathogenesis in the brain after peripheral exposure. Prions are accumulated in the lymphoreticular system (LRS), but the identity of the cells containing infectivity and their role in neuroinvasion have not been determined. We show here that although prion infectivity in the spleen is associated with B and T lymphocytes and to a lesser degree with the stroma, no infectivity could be detected in lymphocytes from blood. In wild‐type mice, which had been irradiated and reconstituted with PrP‐deficient lymphohaematopoietic stem cells and inoculated with scrapie prions, infectivity in the spleen was present in the stroma but not in lymphocytes. Therefore, splenic B and T lymphocytes can either synthesize prions or acquire them from another source, but only when they express PrP.

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia

PrP knockout mice with disruption of only the PrP‐encoding region (Zürich I‐type) remain healthy, whereas mice with deletions extending upstream of the PrP‐encoding exon (Nagasaki‐type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co‐expression of full‐length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N‐terminal tail. We now show that in Zürich I‐type PrP‐null mice, expression of N‐terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.

B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice

Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking. In spleens of wild-type mice, infectivity is associated with B and T lymphocytes and stroma but not with circulating lymphocytes. We generated transgenic prion protein knockout mice overexpressing prion protein in B lymphocytes and found that they failed to accumulate prions in spleen after i.p. inoculation. We conclude that splenic B lymphocytes are not prion-replication competent and that they acquire prions from other cells, most likely follicular dendritic cells with which they closely associate and whose maturation depends on them.