Although it is well-known as a substrate for PrPSc in prion diseases, the normal functions of PrPc in cellular processes and non-prion CNS diseases remain enigmatic. In the EAE model of CNS autoimmunity, we describe that PrPc is an important inhibitor of peripheral immune function. Deletion, knockdown or blockade of PrPc resulted in accelerated and more severe autoimmunity while PrPc overexpression (on a PrP knockout background) resulted in diminished autoimmunity.
Consistent with recently published findings (Tsutsui et al.
; Ingram et al.
), we have found that EAE severity was significantly increased in PrP knockout mice. Based purely on the knockout results, however, it remained unclear whether the effects of PrPc
on CNS autoimmunity were due to alterations in the susceptibility of the CNS to inflammation (i.e. neuronal survival) or due to alterations of the inflammatory response. Additionally, because the knockout animals lack all PrPc
expression, it was not apparent whether alterations in immune function were due to defects in development or thymic negative selection. Pharmacological in vivo
silencing of PrPc
siRNA allowed us to define the role of this molecule more precisely in different disease stages. PrPc
expression was effectively silenced solely in cells of the peripheral immune system but not brain tissue after treatment with Prnp
-siRNA. In addition, Cy3-labelled siRNA was undetectable in CNS tissue indicating that the major location of siRNA knockdown is the periphery. Consequently, we conclude that the major effects of PrPc
in EAE pathogenesis are based on the decrease in PrPc
expression in lymphatic tissues. Furthermore, Prnp-
siRNA administration in adult mice resulted in rapidly enhanced immune responses, thereby bypassing the potential for thymic defects to account for the observed exacerbated autoimmunity. Although we cannot discount the function of PrPc
in the thymic maturation processes in the knockout mouse, it appears that PrPc
disruption in the peripheral immune system is sufficient to enhance immune responses.
The finding that PrPc-silencing could exacerbate disease when delivered at the time of immunization, onset of disease, or at later stages of autoimmunity strongly suggests that PrPc is an important negative regulator of T cell responses.
Our data on in vivo
T cell tracking of cells treated with fluorescent Prnp
-siRNA or nonsense-siRNA strongly suggest that some of the effects we observed in mice treated with Prnp
-siRNA, including the increased number of inflammatory infiltrates in the CNS, may be an indirect effect of Prnp
-silencing on other lymphocyte populations, resulting in an amplification of the initial immune response. These data are complementary to observations we made in our adoptive transfer experiments (E, Supplementary Table S1
F) and in some of the survival experiments. Specifically, Prnp
-siRNA or nonsense-siRNA-transfected purified OVA323–339
TCR transgenic CD45.2+
T cells were transferred intravenously into C57BL/6 CD45.1+
wild-type mice that were immunized with OVA323–339
in complete freund's adjuvant 24 h after cell transfer to investigate whether an effect of PrPc
on the survival of activated antigen-specific T cells may account for some of its effects on CNS autoimmune disease. At Day 5 post transfer, there was no difference with regard to in vivo
antigen-specific proliferation between OVA323–339
TCR transgenic CD4+
T cells transfected with Prnp
-siRNA (B), or with nonsense-siRNA (C). In contrast, silencing of Prnp in vitro
resulted in increased proliferation of bulk T cell populations obtained from splenocytes (A–D).
To study the role of PrPc as a negative regulator of T lymphocyte responses in more detail, we examined the effects of PrPc knockdown on T cell activation and survival.
PrPc is a negative regulator of TCR signalling
We consistently observed an increased antigen-specific proliferation when PrPc was silenced only on T cells, rather than on T cells and antigen-presenting cells. At this point, we are investigating the differential effect of PrPc silencing and overexpression on different cell types. From preliminary data it appears that silencing of PrPc in dendritic cells may be pro-apoptotic, while it is anti-apoptotic in T cells.
TCR signalling is critical to the development of EAE at the immunization and elicitation phases of the disease. Among the earliest events of TCR engagement is recruitment of leukocyte-specific protein tyrosine kinase and subsequent phosphorylation of the TCR zeta chain. Phosphorylated zeta serves as a binding site for ZAP-70 (Chan et al.
), which then phosphorylates numerous substrates including linker of activated T cells. Although it is so far unclear how PrPc
interacts with or influences ZAP-70, PrPc
immunoprecipitated with ZAP-70 in activated T cells in a previous study (Mattei et al.
We found an increased phosphorylation of ZAP-70 in activated T cells after PrPc
silencing. Suppression of the ZAP-70 dependent pathway by PrPc
was confirmed by demonstrating that basal and CD3/CD28-stimulated transcription from NFAT/AP-1, two transcription factors crucial in T cell activation and differentiation, were also enhanced upon silencing PrPc
in T cells (B). Interestingly, NFAT transcription was not altered in the PMA/ionomycin-stimulated T cells. Due to the fact that PMA/ionomycin induces NFAT/AP-1-dependent transcription by activation of protein kinase C/Ras/mitogen-activated protein kinase and calcium mobilization, respectively (Chatila et al.
; Liu and Heckman 1998
), our results support the notion that PrPc
functions as a regulator of TCR-proximal signalling. The in vivo
importance of PrPc
as a negative regulator of TCR signals was demonstrated by the increased incidence of spontaneous EAE in the autoimmune-prone MBP1–11
TCR-transgenic mice after treatment with Prnp
We also demonstrate that PrPc may exert an indirect immunosuppressive function by promoting T cell death. Increased numbers of antigen-specific T cells in vivo after silencing of Prnp allow several explanations: (i) a decreased sequestration of these cells from secondary lymphoid organs into other compartments; (ii) a decrease of Fas-mediated apoptotic cell death; (iii) a diminished growth factor withdrawal due to decreased antigen-specific proliferation or (iv) a push of these cells into cell cycle. The latter scenario was basically ruled out by showing that there was no difference in proliferation of antigen-specific T cells transfected with Prnp-siRNA or nonsense-siRNA. Furthermore our results on increased T cell survival in vitro after silencing of PrPc strongly argue against the hypothesis of diminished growth factor withdrawal from the Prnp-siRNA-treated cells, as more cells were competing for growth factors in a closed system for a 10 day period. These results also rule out differences in tissue sequestration between the two treatment groups. Thus, PrPc may promote apoptosis in activated antigen-specific T cells, possibly mediated through a Fas pathway, or other tumor necrosis factor receptor superfamily member pathways.
Summary and proposed model for PrPc
In summary, we reveal PrPc as a molecule critically influencing T cell functions, by modulating TCR signals and limiting survival. We can thereby clearly assign an essential function to PrPc beyond being the replicative substrate or transport molecule for prions. Whether the conversion from PrPc to the infectious PrPSc conformer also involves changes in T cell function, and whether these have anything to do with prion disease mechanisms is currently unclear. It is possible that the effect for PrPc on regulating TCR signals may suggest a similar function in other cell types. Other cellular receptors, including the B cell receptor, Fc receptor, epidermal growth factor receptor and insulin receptor possess a similar signalling architecture as the TCR. Our observations put PrPc at the intersection of neuroinflammation and neurodegeneration.