Calcium dynamics and vesicle-release proteins in a prion-infected neuronal cell line

Abstract

The prion particle, PrPSc, is an infectious, misfolded form of the cellular prion protein, PrPC that is expressed at high levels in the central nervous system. Prions cause neurodegenerative diseases with clinical signs including dementia, ataxia and myoclonus. These diseases cause characteristic electroencephalographic changes. Neuropathological findings characteristic for prion diseases are spongiform degeneration, astrogliosis and neuronal cell death. Loss of presynaptic proteins such as SNAP-25, syntaxin 1, synaptophysin and synapsin 1 has been observed using immunohistochemistry.

The aim of this thesis was to characterize alterations in presynaptic components implicated in neurotransmission that could be of pathogenetic importance for brain dysfunction in prion diseases. For this purpose I used an immortalized gonadotropin-releasing neuronal cell line (GT1-1) that is susceptible to a mouse-adapted scrapie strain. This cell line represents a suitable system for my studies since the cells possess well characterized voltage-gated ion channels and express several proteins involved in regulated exocytosis.

First, using fluorometric calcium measurements and patch clamp technique, we found reduced voltage-gated N-type calcium currents in scrapie-infected GT1-1 cells whereas the L-type calcium currents were unaffected. No change in the expression of the N-type a1b subunit could be observed indicating that the impairment in N-type calcium channel function does not reside in altered levels of this protein.

Second, using Western blotting we found decreased complex formation of proteins involved in exocytosis, i.e. SNAP-25, syntaxin 1A and synaptophysin. The reduced level of complexed forms of these proteins resulted in an increased amount of monomeric SNAP-25, syntaxin 1A and synaptophysin that could be correlated to the level of PrPSc. However, when complex formation was abolished using exposure of samples to heat, a reduced expression of these monomeric proteins could be revealed in the scrapie-infected GT1-1 cells as compared to uninfected controls. Quinacrine, a compound that has been shown to clear scrapie infection from cell cultures, cleared PrPSc also from the ScGT1-1 cultures and caused a restore, at least partially, of the N-type calcium currents and of the levels of monomeric SNAP-25 and synaptophysin.

Finally, I observed that the reduced complex formation in ScGT1-1 cells was not caused by an increased excitability of the cells. Instead, long-term depolarization could surmount the reduced complex formation.

Taken together, these results indicate that a prion infection can cause functional changes in presynaptic components and possibly electrochemical transmission in neurons, which may contribute to brain dysfunction in prion diseases. Finally, from the perspective of therapeutic strategies the notion that these changes are reversible is of importance.