Prion protein involvement in Reelin/Dab1 signalling: physiological and pathological aspects

Cellular prion protein (PrPC) has been widely investigated since its misfolded isoform, scrapie prion protein (PrPSc), which form aggregates that accumulate in the brain, causes prion diseases. The pathogenesis of prion diseases is currently under debate as they could be gain of toxic function consequences of the formation of PrPSc, or could be due to the loss of the normal physiological function of PrPC. For this reason, a better definition of the exact physiological function of PrPC represents a critical challenge since Prnp knockout mice lack striking phenotype. PrPC is highly abundant in the nervous system where it plays an important role in modulating cell signalling, acting as a dynamic scaffold for the assembly of many different signalling molecules at the neuronal surface. Importantly for the present study, it has been reported that PrPC is able to modulate Fyn, PI3K, AKT/PKB and GSK3β activity. All these intracellular kinases could be also regulated by yet another protein, Reelin, an extracellular matrix glycoprotein that activates a linear downstream signalling pathway. Interestingly, it has been reported that Reelin-signalling pathway is involved in both Alzheimer’s and prion diseases. Indeed, the intracellular adaptor protein Dab1 is able to affect APP processing and intracellular trafficking and to influence amyloid beta deposition in sporadic Creutzfeldt–Jakob disease. In the present study, taking advantage of different mouse models, we analysed the functional interaction between the prion protein and Dab1 signalling cascade from physiological and pathological point of view. In the first part of the project, using a Prnp knockout mouse model, we addressed the question whether PrPC could modulate the Reelin-signalling cascade. If this is the case, we could expect impairments in Reelin downstream signalling when PrPC expression is ablated. Indeed, the expression level and the activation state of Dab1 were evaluated in wild-type (Prnp+/+) and PrPC-null (Prnp0/0) mice brain and primary neuronal cultures. Reelin-conditioned medium, obtained from stably transfected HEK293T cells, was used to stimulate Dab1 activation in vitro and analyse a putative differential activation between the two genotypes. Moreover, recombinant PrP and fusion PrP-hFc proteins were applied in the same system to evaluate the ability of PrPC to stimulate directly the signalling cascade. Dab1 protein resulted upregulated in Prnp0/0 brain in comparison to wild-type animals, with no changes in mdab1 gene transcription levels. Moreover, Prnp0/0 neurons appeared less responsive to Reelin stimulation than controls, leading to reduced phosphorylation of Dab1. Unfortunately, in our conditions, neither recombinant PrP nor PrP-hFc fusion protein were able to induce Dab1 phosphorylation, suggesting a modulating role, instead of a direct effect, of PrPC on the pathway. Expression analysis of different component of the pathway revealed no statistical differences between the two genotypes, with the exception of Fyn kinase, which resulted less phosphorylated in Prnp0/0 mice compared to wild-type controls. Immunoprecipitation experiments failed to unravel an interaction between PrPC and Reelin or its receptor ApoER2, suggesting that impairments in Dab1 expression and phosphorylation are not due to a direct coupling between these proteins at the cell surface. Interestingly, in Prnp0/0 neurons, reduced levels of NCAM protein, a known PrPC interactor, were detected. In the second part of the project, taking advantage of different mouse models of Reelin pathway disruption, we addressed the opposite question to understand whether alteration of the Reelin-signalling pathway could affect the expression of PrPC. However, PrPC expression levels did not resulted significantly different in comparison to their respective controls in none of the genotypes analysed. These results suggest that, while PrPC is able to modulate Reelin-signalling cascade and in particular Dab1 activation state, alterations of this pathway do not affect PrPC expression. Finally, terminally sick mice intracerebrally inoculated with RML prion strain were used as prion pathology model. Interestingly, in terminal stages of prion infection, Dab1 and Reelin receptors signals are no longer detectable. NCAM expression level seems not affected by prion infection, while Fyn kinase and AKT levels are reduced by 30% and 50% respectively. Total Reelin amount is not modified by prion infection, while full-length protein is reduced by almost 50% in RML-infected samples compared to controls with a concomitant 2-fold increase of both cleavage products. Taken together, these findings suggest the existence of a functional interplay between prion protein and Dab1 signalling. Results obtained using PrPC-null mice as model of loss-of-function indicate a role for PrPC in promoting Dab1 signalling. However, as Reelin and its receptors are not affected by Prnp ablation, more likely PrPC indirectly modulate Dab1 signalling through NCAM and Fyn pathway. Interestingly, Dab1 signalling resulted completely abolished in RML-inoculated terminally sick mice, used as prion disease model, together with strong alteration in Reelin processing, already reported in Alzheimer’s disease patients. Further experiments are in progress in order to clarify the mechanisms by which PrPC and PrPSc modulates Dab1 signalling pathway.