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Previous studies have shown that cellular prion protein (PrP(C)) plays anti-apoptotic and antioxidative role against cell death induced by serum-deprivation (SDP) in an immortalized prion protein gene-deficient neuronal cell line derived from Rikn prion protein (PrP) gene-deficient (Prnp(-/-)) mice, which ectopically produce excess Doppel (Dpl) (PrP-like glycoprotein). To investigate whether PrP(C) inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp(-/-) mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp(-/-) cell line (NpL2) showed that PrP(C) potently inhibited SDP-induced apoptotic cell death. Furthermore, PrP(C) expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti-apoptotic and anti-oxidative functions of PrP, suggesting that PrP(C) may be directly correlated with protection against oxidative stress.

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL3-4 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL3-4 cells expressing Dpl fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.

The Prnd-encoded prion protein (PrP)-like protein, Doppel (Dpl), is a homologue of Prnp-encoded PrP, and is N-glycosylated protein with glycosylphosphatidylinositol anchor like PrP. Recently, ectopic expressions of Prnp/Prnd chimeric mRNAs have been identified as the cause of late-onset ataxia observed in several lines of Prnp-knockout mice such as ZrchII, Ngsk, Rcm0, and Rikn mice. However, it remains unclear whether the toxic effect of Dpl expression is a cell-autonomous mechanism but rather reflect a systemic process of heterogeneous cell population in the brain. In this study, the cell-autonomous role of Dpl was estimated by investigating PrP-deficient cells (HpL3-4)-the SV40 large T-antigen immortalized and Rikn Prnp(-/-) mice-derived neuronal cell line expressing Prnp/Prnd chimeric mRNAs. The reverse transcription polymerase chain reaction revealed that serum deprivation did not increase Prnp/Prnd chimeric mRNAs, which in fact was translated into a small amount of Dpl in HpL3-4 cells, whereas serum deprivation induced apoptotic cell death of HpL3-4 cells. Dpl overexpression enhanced apoptotic cell death, whereas the toxic effect of Dpl on apoptotic cell death was neutralized by PrP expression. These results indicate that Dpl elicited dose-dependently toxic effects on PrP-deficient cells without affecting on PrP-expressing cells, suggesting that the PrP-Dpl interaction can regulate cell death in a cell-autonomous manner.

The function of cellular prion protein (PrPC), which is a copper binding protein, remains unclear. To elucidate the mechanisms in which PrPC is involved in neuroprotection, we compared death signals in prion protein gene-deficient (Prnp-/-) primary cerebellar granular neurons (CGNs) to those with wild-type (WT) CGNs. When copper was exposed to these CGNs, ZrchI, and Rikn Prnp-/- CGNs were more sensitized and underwent apoptotic cell death more readily than WT CGNs. Furthermore, the level of intracellular hydrogen peroxide (H2O2) in WT CGNs increased by copper toxicity, whereas those in ZrchI and Rikn Prnp-/- CGNs did not. These results suggest that PrPC modulates the intracellular H2O2 level as a copper-binding protein to protect CGNs from apoptotic cell death possibly due to inhibiting a Fenton reaction., (Copyright 2004 Elsevier Inc.)

The protective effect of synthetic serum thymic factor (FTS) nonapeptide on lipopolysaccharide (LPS)-induced pancreatic cell damage in 10-week-old BALB/c male mice was investigated. Mice were divided into three groups. Group I was treated with LPS (10 micro g/head; i.p.) (LPS-treated mice). Group II was administered with FTS (50 micro g/head; i.p.) 24 hr before treatment with LPS and complemented immediately before LPS injection with FTS (50 micro g/head; i.p.) (FTS-administered mice). Group III was only treated with the same volume of saline (control mice). Treatment of LPS in vivo resulted in the destruction of pancreatic acinar cells. In those cells, many apoptotic cells were detected by immunohistochemistry using an anti-single stranded DNA antibody. Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) revealed that LPS treatment also caused low or a lack of insulin expression in pancreatic islets. In contrast, morphological change was not seen and apoptotic cell death was suppressed in pancreatic cells of FTS-administered mice. Moreover, insulin expression was normal in those mice. FTS administration enhanced expression of Bcl-2 mRNA levels in pancreatic tissues and IL-6 mRNA levels in splenocytes significantly compared with those of LPS treatment at 3 hr after LPS injection. These findings suggest that FTS prevents LPS-induced cell damage via enhancing Bcl-2 expression in the pancreas and systemic IL-6 production.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine considered to play a key role in beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). Serum thymic factor (Facteur thymique serique; FTS) is a nonapeptide thymus hormone known to inhibit IDDM in a mouse model. In this study, the effect of TNF-alpha on the murine pancreatic beta-cell line MIN6 was examined. Cell shrinkage and detachment were seen in cells treated with 0-50 ng/ml TNF-alpha for 12h. Oligonucleosomal DNA fragmentation was determined from non-adherent cells, indicating that the TNF-alpha-induced cell destruction was attributed to apoptosis. Fragmented DNA was quantified by enzyme-linked immunosorbent assay to measure the amount of histone-bound oligonucleosomes. FTS was treated with TNF-alpha and the percentage of fragmented DNA was analyzed. The data indicate a distinct reduction of fragmented DNA at a concentration of 1 ng/ml FTS. Expression of TNF receptor I, inducible form of nitric oxide synthase (iNOS), interleukin-1 beta-converting enzyme (ICE), Bcl-2, and nuclear factor kappa B (NF-kappa B) was analyzed by reverse transcriptase-polymerase chain reaction to investigate the suppressor mechanism of FTS on TNF-alpha-induced apoptosis. FTS treatment suppressed the expression of iNOS and Bcl-2 mRNA in TNF-alpha-treated cells. The expression of NF-kappa B mRNA in TNF-alpha-treated cells was enhanced after FTS treatment, while that of ICE mRNA did not change in TNF-alpha-treated cells with or without FTS treatment. These results suggest that the inhibition of MIN6 cell death by FTS on TNF-alpha-induced apoptosis is caused by a negative feedback mechanism involving the inhibition of iNOS induction.

Prion protein gene (Prnp)-deficient(Prnp(-/-)) neuronal cells are more susceptible to serum deprivation compared to Prnp(+/+) neuronal cells. However, little is known about the cell death of Prnp(-/-) neuronal cells under serum deprivation. In this study, as a known neuroprotective agent we analyzed the effect of tumor necrosis factor-alpha (TNF-alpha) on the cell death of Prnp(-/-) neuronal cells. Although expression of Bcl-2 and Bcl-x(L) decreased in a time-dependent manner under serum deprivation, treatment with TNF-alpha protected Prnp(-/-) neuronal cells from serum deprivation with an increase in anti-apoptotic proteins Bcl-2 and Bcl-x(L). Nuclear morphological analysis using fluorescence microscopy and flow cytometry analysis showed that gene transfer of bcl-2 or bcl-x(L) significantly inhibited apoptosis induced by serum deprivation. These findings indicate that TNF-alpha attenuated cell death of Prnp(-/-) neuronal cells by induction of Bcl-2 and Bcl-x(L),and that decreases in Bcl-2 and Bcl-x(L) played crucial roles in the apoptosis of Prnp(-/-) neuronal cells.

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.

Abstract: Splenocytes of wild-type (Prnp +/+) and prion protein gene-deficient (Prnp −/−) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA)+ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrPC) expression was enhanced following ConA stimulation, but not PMA+Io or LPS in Prnp +/+ splenocytes. Rikn Prnp −/− splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp −/− splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrPC and PrPLP/Doppel. [Copyright &y& Elsevier]

Abstract: Cellular prion protein (PrPC) plays anti-apoptotic and anti-oxidative roles in apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line. The octapeptide repeat region (OR) and N-terminal half of the hydrophobic region (HR) of PrPC are indispensable for PrPC activity, but the mechanisms remain unclear. In the present study, elucidation of the mechanisms by which PrPC elicits the anti-oxidative activities was facilitated by evidence of stress-inducible protein 1 (STI1) mediating PrPC-dependent superoxide dismutase (SOD) activation. Immunoprecipitation revealed that PrPC was associated with STI1. The inhibitory peptides against PrPC–STI1 binding [STI1 pep.1 and PrP(113–132)] indicated toxic activity in PrPC-expressing cells by inhibiting SOD activity but not in Prnp−/− cells. Furthermore, OR and N-terminal half of the HR were required for the inhibitory effect of PrP(113–132) but not STI1 pep.1. These data are consistent with results established with a model where OR and N-terminal half of the HR mediate the action of STI1 upon cell survival and upregulation of SOD activity. [Copyright &y& Elsevier]

Abstract: Cellular prion protein PrPC contains two evolutionarily conserved domains among mammals; viz., the octapeptide repeat region (OR; amino acid residue 51–90) and the hydrophobic region (HR; amino acid residue 112–145). Accumulating evidence indicates that PrPC acts as an inhibitor of apoptosis and regulator of superoxide dismutase (SOD) activity. To further understand how PrPC activates SOD and prevents apoptosis, we provide evidence here that OR and N-terminal half of HR mediate PrPC-dependent SOD activation and anti-apoptotic function. Removal of the OR (amino acid residue 53–94) enhances apoptosis and decreases SOD activity. Deletion of the N-terminal half of HR (amino acids residue 95–132) abolishes its ability to activate SOD and to prevent apoptosis, whereas that of the C-terminal half of HR (amino acids residue 124–146) has little if any effect on the anti-apoptotic activity and SOD activation. These data are consistent with a model in which the anti-apoptotic and anti-oxidative function of PrPC is regulated by not only OR but also the N-terminal half of HR. [Copyright &y& Elsevier]