SARM1 participates in axonal degeneration and mitochondrial dysfunction in prion disease.

Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss, axonal degeneration, and mitochondrial dysfunction. Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1. We found that depletion or dysfunctional mutation of SARM1 protected against NAD+ loss, axonal degeneration, and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126. NAD+ supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect. NAD+ supplementation in PrP106-126-incubated N2a cells, SARM1 depletion, and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival. Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP106-126 are partially dependent on SARM1 NADase activity. This pathway has potential as a therapeutic target in the early stages of prion disease.

PRAS40 alleviates neurotoxic prion peptide-induced apoptosis via mTOR-AKT signaling.

AIMS: The proline-rich Akt substrate of 40-kDa (PRAS40) protein is a direct inhibitor of mTORC1 and an interactive linker between the Akt and mTOR pathways. The mammalian target of rapamycin (mTOR) is considered to be a central regulator of cell growth and metabolism. Several investigations have demonstrated that abnormal mTOR activity may contribute to the pathogenesis of several neurodegenerative disorders and lead to cognitive deficits. METHODS: Here, we used the PrP peptide 106-126 (PrP106-126 ) in a cell model of prion diseases (also known as transmissible spongiform encephalopathies, TSEs) to investigate the mechanisms of mTOR-mediated cell death in prion diseases. RESULTS: We have shown that, upon stress caused by PrP106-126 , the mTOR pathway activates and contributes to cellular apoptosis. Moreover, we demonstrated that PRAS40 down-regulates mTOR hyperactivity under stress conditions and alleviates neurotoxic prion peptide-induced apoptosis. The effect of PRAS40 on apoptosis is likely due to an mTOR/Akt signaling. CONCLUSION: PRAS40 inhibits mTORC1 hyperactivation and plays a key role in protecting cells against neurotoxic prion peptide-induced apoptosis. Thus, PRAS40 is a potential therapeutic target for prion disease.

Overexpression of BAT3 alleviates prion protein fragment PrP106-126-induced neuronal apoptosis.

BACKGROUNDS AND AIMS: Prion diseases are a group of infectious neurodegenerative diseases characterized by neuronal death and degeneration. Human leukocyte antigen-B-associated transcript 3 (BAT3) is an important apoptosis regulator. We therefore investigated the interactions between BAT3 and prion protein and the potential role of BAT3 in PrP106-126-induced apoptosis. METHODS: BAT3 and prion protein were overexpressed in Hela, Neuro2A, or primary neuronal cells by transfection with BAT3-HA or PRNP-EGFP expression plasmids and their relationship studied by immunofluorescence and Western blotting. The effect of BAT3 on PrP106-126-induced cytotoxicity and apoptosis was detected by the CCK-8 assay and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The expression of cytochrome c and Bcl-2 was examined by Western blotting. RESULTS: BAT3 interacted with prion protein and enhanced PrP expression. After PrP106-126 peptide treated, BAT3 was transported from the nucleus to cytoplasm, increased cell viability, and protected neurons from PrP106-126-induced apoptosis through stabilizing the level of Bcl-2 protein and inhibiting the release of cytochrome c to cytoplasm. CONCLUSIONS: Our present data showed a novel molecular mechanism of PrP106-126-induced apoptotic process regulation through the overexpression of BAT3, which may be important for the basic regulatory mechanism of neuron survival in prion diseases and associated neurodegenerative diseases in vivo.

Hsp70 binds to PrPC in the process of PrPC release via exosomes from THP-1 monocytes.

PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the surface of a number of peripheral blood cells. PrPC must be present for the generation and propagation of pathogenic conformer [PrPSc (scrapie prion protein)], which is a conformational conversion form of PrPC and has a central role in transmissible spongiform encephalopathies. It is important to determine the transportation mechanism of normal PrPC between cells. Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. We have identified that THP-1 monocytes can secrete exosomes to culture medium, and the secreted exosomes can bear PrPC. We also found that Hsp70 interacts with PrPC not only in intracellular environment, but in the secreted exosomes. However, the specific markers of exosomes, Tsg101 and flotillin-1, were found with no interaction with PrPC. Our results demonstrated that PrPC can be released from THP-1 monocytes via secreted exosomes, and in this process, Hsp70 binds to PrPC, which suggests that Hsp70 may play a potential functional role in the release of PrPC.

Polymorphism of prion protein gene in sheep of Inner Mongolian, China.

Susceptibility to natural scrapie in sheep is associated with polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene. To assess the risk of scrapie in sheep raised in China, DNA from 30 sheep of two breeds was isolated, amplified and sequenced for the PrP gene. The ovine PrP gene was found to be highly homogenous. The genotype associated with high susceptibility to scrapie (VRQ) was absent, whereas that associated with the resistance (ARR) was present in 6.7% of sheep examined. ARK was also rare (6.7%). ARQ that is associated with an intermediate susceptibility was the genotype observed in the most of sheep examined (86.6%). These data suggest that Chinese sheep of Mongolian sheep breed are susceptible to scrapie.

PrP 106-126 altered PrP mRNA gene expression in mouse microglia BV-2 cells.

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126, and indicate that microglial cells might play a critical role in prion pathogenesis.

Variable levels of 37-kDa/67-kDa laminin receptor (RPSA) mRNA in ovine tissues: potential contribution to the regulatory processes of PrPSc propagation?

The 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR, also known as ribosomal protein SA, RPSA) has been reported to be involved in cancer development and prion internalization. Previous studies have shown that the LRP/LR is expressed in a wide variety of tissues. In particular, expression of LRP/LR mRNA may be closely related to the degree of PrP(Sc) propagation. This study presents a detailed investigation of the LRP/LR mRNA expression levels in eleven normal ovine tissues. Using real-time quantitative PCR, the highest LRP/LR expression was found in neocortex (p < 0.05). Slightly lower levels were found in the heart and obex. Intermediate levels were seen in hippocampus, cerebellum, spleen, thalamus, mesenteric lymph node, and the lowest levels were present in liver, kidney, and lung. In general, the LRP/LR mRNA levels were much higher in neuronal tissues than in peripheral tissues. The observation that differences in LRP/LR mRNA expression levels are consistent with the corresponding variation in PrP(Sc) accumulation suggests that the 37-kDa/67-kDa laminin receptor may be involved in the regulation of PrP(Sc) propagation.

Copper(II) inhibits in vitro conformational conversion of ovine prion protein triggered by low pH.

To gain insight into the conformational conversion of ovine prion protein (OvPrP(C)) at different pH values and/or in the presence of CuCl(2), the secondary structure of OvPrP(C) was analysed by circular dichroism (CD) spectroscopy. Copper treatment of OvPrP(C) under moderately acidic conditions (pH approximately 5.0-6.0) as well as physiological conditions (pH 7.4) also makes OvPrP(C) adopt protease-resistant and beta-sheet-rich conformation. However, under lower pH conditions (2.0-4.5) with copper treatment, OvPrP(C) gained higher alpha-helix structure. This study demonstrated that Cu(2+) can significantly modulate conformational conversion triggered by acidic pH, and this will provide therapeutic intervention approaches for prion diseases.