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BAVARIAN RESEARCH ASSOCIATION PRIONS

LMU 10 Pathophysiological role of prion proteins at hippocampal excitatory synaptic terminals

Field of work:

Aim: With this study we aimed to understand basic mechanisms of synaptic dysfunction in prion diseases as a major substrate of neurological impairment in patients suffering from Creutzfeldt-Jakob disease. Rationales and Experimental approaches: First we analysed the neuronal calcium homeostasis and calcium dependent membrane currents in acute brain slice preparation of mice lacking PrP. We chose to analyse the cytosolic calcium homeostasis because our previous studies on synaptic transmission in Prnp-/- mice indicated that synaptic transmission is impaired due to alterations in intracellular calcium. Second we aimed to analyse the functional role of the copper binding N-terminal domain of PrP by studying cerebellar granule cells derived from transgenic mice overexpressing Nterminal deleted PrP on the one hand and immortalized Prnp-/- cells transfected with different mutant-PrP constructs on the other hand. Third we monitored the time course of synapse degeneration in scrapie infected mice throughout the disease. Here we analysed the degeneration of dendritic spines, the side of excitatory synapses, in the alive scrapie infected animal applying in vivo 2-photon imaging. Results and Discussion: I) Acute hippocampal brain slices from Prnp-/- mice: Patch clamp measurements revealed that the calcium dependent slow afterhyperpolarisation current (sIAHP) is reduced in hippocampal neurons lacking PrP. Confocal measurements of the cytosolic calcium concentration revealed that this alteration is most likely due to a reduced calcium influx through voltage gated calcium channels (VGCCs) and consecutive a reduced calcium release from intracellular calcium stores. Indeed further Patch-clamp studies on VGCCs revealed a reduced influx. Western-Blots showed that this is most likely due to an alteration of the expression of the VGCC beta2 subunits (Fuhrmann et al. 2006), most likely as a consequence of a reduced phosphatidylinositol 3-kinase activity (see below). IIa) Immortalized hippocampal neurons derived from Prnp-/- mice transfected with PrPdeletion mutants: Biochemical studies revealed a functional link between PrPC expression and phosphatidylinositol 3-kinase (PI3-kinase) activation, a protein that plays a pivotal role in cell survival. Moreover, both PI3-kinase activation and cytoprotection by PrPC expression appeared to rely on copper binding to the N-terminal octapeptide of PrPC (Vassallo et al. 2005). Since PI3-kinase modulates the expression of the beta2 subunit of VGCC we suppose that the alteration in the calcium homeostasis (see I) are the consequence of an alteration in PI3-kinase activity. IIb) Cultured cerebellar granule cells from Prnp-/- mice and transgenic mice overexpressing Nterminally deleted PrPC: Calcium imaging studies revealed that PrPC modulates the release from intracellular calcium stores in response to hydrogen peroxide. Moreover, the N-terminal octapeptide repeats of PrPC were essential for this phenotype, because neurons derived from transgenic mice expressing a PrPC that lacked the N-terminus resembled the PrPC-deficient phenotype. The results indicate that PrPC might function as a sensor for H2O2 thereby activating a protective signalling cascade involving kinases like fyn and PI3-kinase (Krebs et al. 2006). III) In vivo two-photon microscopy of excitatory synapses in scrapie infected mice: 2-photon in vivo imaging of cortical dendrites of excitatory neurons over two months revealed a linear decrease of spine density. Interestingly, only persistent spines (lifetime ≥ 8 days) disappeared, while the density of transient spines (lifetime ≤ 4 days) was unaffected. Prior to spine loss, dendritic varicosities emerged preferentially at sites where spines protrude from the dendrite. These results implicate that the location where the spine protrudes from the dendrite may be particularly vulnerable and that dendritic varicosities may actually cause spine loss (Fuhrmann et al. revised submission J. Neurosci).