Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.

The serotonergic system plays important roles in multiple functions of the nervous system and its malfunctioning leads to neurological and psychiatric disorders. Here, we show that the cell adhesion molecule close homolog of L1 (CHL1), which has been linked to mental disorders, binds to a peptide stretch in the third intracellular loop of the serotonin 2c (5-HT2c) receptor through its intracellular domain. Moreover, we provide evidence that CHL1 deficiency in mice leads to 5-HT2c-receptor-related reduction in locomotor activity and reactivity to novelty, and that CHL1 regulates signaling pathways triggered by constitutively active isoforms of the 5-HT2c receptor. Furthermore, we found that the 5-HT2c receptor and CHL1 colocalize in striatal and hippocampal GABAergic neurons, and that 5-HT2c receptor phosphorylation and its association with phosphatase and tensin homolog (PTEN) and β-arrestin 2 is regulated by CHL1. Our results demonstrate that CHL1 regulates signal transduction pathways through constitutively active 5-HT2c receptor isoforms, thereby altering 5-HT2c receptor functions and implicating CHL1 as a new modulator of the serotonergic system.

Prion protein (PrP) plays crucial roles in regulating antioxidant systems to improve cell defenses against cellular stress. Here, we show that the interactions of PrP with the excitatory amino acid transporter 3 (EAAT3), c-glutamyl transpeptidase (c-GT), and multi-drug resistance protein 1 (MRP1) in astrocytes and the interaction between PrP and EAAT3 in neurons regulate the astroglial and neuronal metabolism of the antioxidant glutathione. Ablation of PrP in astrocytes and cerebellar neurons leads to dysregulation of EAAT3-mediated uptake of glutamate and cysteine, which are precursors for the synthesis of glutathione. In PrP-deficient astrocytes, levels of intracellular glutathione are increased, and under oxidative stress, levels of extracellular glutathione are increased, due to (i) increased glutathione release via MRP1 and (ii) reduced activity of the glutathione-degrading enzyme c-GT. In PrPdeficient cerebellar neurons, cell death is enhanced under oxidative stress and glutamate excitotoxicity, when compared to wild-type cerebellar neurons. These results indicate a functional interplay of PrP with EAAT3, MRP1 and c-GT in astrocytes and of PrP and EAAT3 in neurons, suggesting that these interactions play an important role in the metabolic cross-talk between astrocytes and neurons and in protection of neurons by astrocytes from oxidative and glutamateinduced cytotoxicity.