Functional role of cellular prion protein in regulating cell adhesion molecule associated transport systems under physiological and pathophysiological conditions
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The pathogenic form of prion protein (PrPsc) plays a crucial role in the development of spongiform encephalopathies and several neurological disorders in humans, such as Creutzfeld-Jakob disease, Gerstman-Sträussler-Scheinker syndrome and fatal familial insomnia. The cellular form of PrP (PrPc) has protective functions against oxidative stress, hypoxia, ischemia, excitotoxicity or hypoglycemia and loss of PrPc functions lead to neuronal cell death and contribute to the development of severe neurodegenerative disease, such as Parkinson’s or Alzheimer´s disease. In addition, PrPc beneficially sustains neurotransmitter systems, in particular the serotonergic and dopaminergic systems, which are negatively affected by PrPsc. The proposed role of PrPc in the serotonergic and dopaminergic systems by regulating the transport of tryptophan and tyrosine, the precursor for serotonin and dopamine synthesis, could not be confirmed in this study. However, a regulation of the serotonin uptake in astrocytes by PrPc was observed. This observation and the finding that serotonin levels are altered in a gender-specific manner in different brain areas of PrP-deficient mice implies that PrP plays an important role in the serotonin system and thus in the homeostasis of emotional behavior. Lactate released from astrocytes is used by neurons as the main energy source under ischemic or hypoxic conditions. Lactate transport in astrocytes is coupled to α2/β2-Na+/K+-ATPase and regulated by PrPc via its octarepeat region. By a label-free binding assay using synthetic peptides comprising extracellular loop of the α2/β2-Na+/K+-ATPase we revealed strong binding of the loops 1 and 4, but not loops 2 or 3, with recombinant PrP fused to a human Fc, indicating that PrP interacts via these loops to regulate α2/β2-Na+/K+-ATPase activity and α2/β2-Na+/K+-ATPase-coupled lactate transport. Glutamate and cysteine are required for synthesis of glutathione which plays a crucial role in detoxification of reactive oxygen species and xenobiotics. Astrocytes, but not neurons release glutathione into the extracellular space. For glutathione synthesis, neurons depend on extracellular cysteine which is generated from glutathione by the astroglial ectoenzyme γ-glutamyl transpeptidase and the neuronal ectopeptidase. By transfection of CHO cells with the excitatory amino acid transporters EAAC1, Glt-1 or Glast and co-transfection of each transporter with wild-type PrPc or PrPc mutant lacking the octarepeat region, we showed that PrPc specifically regulates uptake of glutamate and cysteine via the EAAC-1. The glutamate transport, but not the cysteine transport depends on the octarepeat region. However, the H2O2-induced cysteine uptake via EAAC-1 depends on the octarepeat region. Under normal and oxidative stress conditions, PrP-deficient astrocytes exhibited alterations in the intracellular and extracellular levels of total, oxidized and reduced glutathione in comparison to wild-type astrocytes. The enhanced extracellular level of reduced glutathione in PrP-deficient astrocytes correlates with a reduced activity of γ-glutamyl transpeptidase, indicating that PrPc regulates the activity of this gluthatione-degrading enzyme. By application of an inhibitor of the multidrug protein 1 (MDR-1), which mediates the export of reduced and oxidized glutathione from astrocytes, we showed that the regulation gluthatione levels by PrP under oxidative stress depends on MDR-1. We showed that PrP-deficient neurons are more susceptible to hypoxia or oxidative stress than wild-type neurons. Moreover, wild-type neurons in co-cultures with wild-type astrocytes showed better survival than in co-cultures with PrP-deficient astrocytes, suggesting that PrP on astrocytes plays a crucial role for the protection of neurons against oxidative stress and ischemic/hypoxic conditions. Furthermore, survival of wild-type neurons upon H2O2 treatment was improved when incubated with conditioned medium from wild-type astrocytes, but not in the presence of medium from PrP-deficient astrocytes. Survival of wild-type cerebellar neurons was also improved upon addition of isolated exosomes which are small membrane vesicles released from cells and which play an important role as vehicle for the propagation of PrPsc and in astrocyte-neuron communication. In particular, exosomes isolated from wild-type astrocytes upon H2O2-treatment or after exposure to hypoxia have pronounced neuroprotective properties. Exosome-free conditioned media have only a minor effect on neuronal survival, underscoring the relevance of exosomes in neuroprotection. Exposure of wild-type astrocytes to hypoxia and reoxygenation leads to an increase in PrP levels in isolated exosomes. Proteome analysis of exosomes from wild-type and PrP-deficient astrocytes under stressed and non-stressed conditions by semi-quantitative mass spectrometry showed differences in protein patterns for exosomes from PrP-deficient astrocytes relative to exosomes from wild-type astrocytes as well as for exosomes from treated astrocytes relative to exosomes from untreated astrocytes. For example, clusterin and apolipoprotein E which are associated with the pathogenesis of prion diseases display different levels: clusterin was only found in exosomes from wild-type astrocytes under normoxic conditions and the level of apolipoprotein E was enhanced in exosomes from wild-type astrocytes exposed to reoxygenation.
