CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

The physiology of the prion protein

Prions are the atypical infectious agents responsible for the onset and propagation of transmissible spongiform encephalopathies, or prion diseases, which are fatal to both animals and man. Within the framework of the "protein only" hypothesis, prions are composed of a conformationally abnormal isoform of the cellular prion protein, PrP, a highly conserved cell surface sialoglycoprotein expressed in all tissues. Although the involvement of PrP in prion diseases is well established, both the mechanism of prion-related neurotoxicity and the physiologic role of the protein remain obscure. The extensive research on the latter issue, almost entirely devoted to the nervous tissue, or to cells of neuronal origin, has not resulted in a proposition recapitulating the multiple, sometime contrasting, observations for the biological role of PrP. Intriguingly, not even the genetic approach has solved the issue, as PrP-knockout animals present no overt phenotype. This finding may support the possibility of a hidden PrP-/- phenotype that could become apparent only under stress conditions. Clearly, the still un-recognised function for PrP demands that alternative approaches be devoted to this issue. In light of these premises, the present laboratory has started a multifaceted project on the functional analysis of PrP using paradigms unprecedented in the prion field, namely post-ischemic perfusion of isolated hearts, regeneration of injured skeletal muscles, and local Ca2+ movements in primary cerebellar granule cells. Importantly, the project has exploited the availability of congenic murine lines that express different PrP amounts, i.e. wild-type FVB and PrP-KO (F10) mice, as well as a line that over-expresses the protein 4 times the normal level (PrP-OE Tg37).

Study of the anti-oxidant and cell-protective role of PrP using an ex-vivo heart model

Within the many functions ascribed to PrP, it has been suggested that the protein serves in the cell defence against oxidative injury. Since PrP is abundantly expressed also in the cardiac muscle, in this line of research we have aimed at verifying the anti-oxidant attribute of PrP. This was achieved by subjecting isolated hearts to ischemia and reperfusion protocols, given that the explosive ROS production during the post-ischemic reperfusion is well known to trigger oxidative damage and death of cardiomyocytes. Thus, ex-vivo hearts from 3 month-old male (WT, F10 and Tg37) mice have been perfused in the non-recirculating Langendorff reverse mode that allows collection of the perfusate for subsequent analyses. To address more specifically the anti-oxidant potential of PrP, the ischemic preconditioning protocol (IPC) (which produces beneficial ROS amounts before ischemia/reperfusion), and the non-ischemic oxidative injury (perfusion in the presence of H2O2), have also been applied.

 

The results obtained so far all concur to strengthen the involvement of PrP in the cellular antioxidant defense. Indeed, (i), over-expressed PrP protected the myocardium against ischemia-reperfusion damage, as evaluated by the lactic dehydrogenase (LDH) release in the coronary effluent (Fig.1), the extent of oxidised myofibrillar proteins (by tropomyosin immunolabeling), and the quantification of ROS accumulation (staining of heart cryosections with dihydroethidine); (ii), these same parameters showed that normal and OE amounts of PrP protected against the oxidative injury induced by perfusion with H2O2; (iii), an inverse relation was found to exist between the content of PrP and the protection elicited by the IPC protocol, as expected were indeed PrP opposing ROS accumulation.

Study of the role of PrP in cell differentiation, using a skeletal muscle degeneration/regeneration model

Using in vitro and whole animal approaches, the contribution of PrP in neurogenesis and differentiation of the central nervous system has recently been proposed. Thus, also because previous observations of our laboratory had shown that PrP is involved in the in vitro differentiation of myocytes, the second line of research has focussed on an in vivo paradigm of PrP role in myogenesis. More specifically, we first degenerated the hind-limb Tibialis anterior (TA) muscle - of 3 month-old male WT, PrP-KO and PrP-OE mice - with a myotoxin (i.e., cardiotoxin, CTx), and then we followed the muscle regeneration (up to 60 days after treatment) using several histologic and biochemical parameters. These included the dimension and maturation (i.e. fusion of myoblasts to form polynucleated cells) of fibres, and the expression of transcriptional factors and/or muscle maturation markers. This study has shown that, compared to animals expressing PrP, recovery from damage was significantly slower in PrP-KO mice. This suggests that PrP plays a role in the morphogenesis of tissues other than the central nervous system, and that this may also occur in the adult life.

 

Indeed, not only we have found that the cross-section area and the fusion index of fibres had a smaller value in the absence of PrP (at 9 and 16 days post injury), but also that the expression of neo-MHC (Fig 2), a late marker of the differentiation process, was significantly less abundant in the PrP-KO strain, although it persisted for a longer period of time than in the presence of PrP. Importantly, we have also shown that these events most likely originated in the very early phases of the muscle repair process, given that, in PrP-less regenerating fibers, both the incorporation by cycling myoblasts of the tymidine analogue 5-bromo-2-deoxyuridine, and the expression of Pax7 (a transcriptional factor marker of the muscle progenitors, satellite cells) had values suggesting that these cells started to proliferate later, or replicated for a longer period of time, than in the presence of PrP.

Study of the involvement of PrP in the control of cell calcium homeostasis

Several lines of evidence have suggested that PrP takes part in multi-component signaling complexes at the cell surface, and that these interactions elicit signals to the cell. The molecular pathway(s) implicated in these events is(are), however, not yet fully understood. In a previous work of ours, we had shown that in a cell model system PrP was controlling local Ca2+ movements. This fact, and the possibility that Ca2+ could be involved in some of the different functions attributed to PrP, have prompted us to study Ca2+ homeostasis in primary cultured cerebellar granule cells (CGC). To accomplish this part of the project - aimed at relating the PrP genotype to Ca2+ homeostasis - comparative analyses have been carried out in CGC derived from WT and PrP-KO mice, following their infection with lentiviral vectors encoding the Ca2+-sensitive aequorin chimerae targeted to cytosolic microdomains of the plasma membrane, the lumen of the endoplasmic reticulum (ER), or the mitochondrial matrix. Ca2+ movements have been recorded after applying appropriate stimuli that either promoted the entry of Ca2+ from the extracellular space, or its release from intracellular stores.

 

This study has confirmed in primary neurons the capacity of PrP to control local Ca2+ movements, as the absence of PrP induced a higher Ca2+ influx from the extracellular space (Fig. 3), as well as a higher Ca2+ replenishment of the mitochondrial matrix, also when Ca2+ was released from the ER. These data are in line with the higher excitability of PrP-KO neurons and the anti-apoptotic property reported for PrP, respectively. Intriguingly, we have also found that the presence of PrP was related to the up-regulation of several proteins involved in the control of Ca2+ homeostasis, e.g., the plasma membrane and the ER Ca2+ ATPases, and ORAI2, a component of store-activated Ca2+ channels.

Publications

  • Sorgato MC, Peggion C, Bertoli A (2009) Is, indeed, the prion protein a Harlequin servant of "many" masters? Prion 3:202-5.
  • Massimino ML, Ferrari J, Sorgato MC, Bertoli A (2006) Heterogeneous PrPC metabolism in skeletal muscle cells. FEBS Lett. 580:878-84.
  • Brini M, Miuzzo M, Pierobon N, Negro A, Sorgato MC (2005) The prion protein and its paralogue Doppel affect calcium signaling in Chinese hamster ovary cells. Mol. Biol. Cell 16:2799-808.

Grants

The Italian Ministero dell'Università e della Ricerca (Prin 2006, to M.C.S)
The University of Padova (Progetto d'Ateneo CPDA089551, to A.B.)

Collaborations

Laboratory of Prof. F. Di Lisa, Dept of Biosciences, Univ. of Padova

 

PI photo

Maria Catia Sorgato

Contact information

email  E-mail

email  049 827 6136

Participating staff

Maria Lina Massimino
Assistant Professor

Alessandro Bertoli
Assistant Professor

Caterina Peggion
Post-doc

Cristian Lazzari
PhD student

Roberto Stella
PhD student

Filippo Zanetti
PhD student