Hypoxia-inducible factor-1 alpha regulates prion protein expression to protect against neuron cell damage – neurobiology of aging brain anoxia

Fig. 1

Hypoxia suppresses prion protein fragment [prp (106–126)]-induced cell death. (A) SH-SY5Y cells were incubated with 200 μm prp (106–126) for 24 hours following growth for 12 hours in normoxic conditions (21% oxygen tension) or hypoxic conditions (1% oxygen tension). Treated cells were photographed with a light microscope (×200). (B) cell viability was measured by an established crystal violet staining method. Viability of control cells was set at 100%, and viability relative to control is presented. (C) analysis of genomic DNA isolated from SH-SY5Y cells treated as described in (A). Marker: 100 base pair DNA ladder. (D) SH-SY5Y cells were treated with the indicated concentrations (μm) of prp (106–126) for 24 hours after exposure to the aforementioned normoxic or hypoxic conditions for 12 hours.Brain anoxia


release of lactate dehydrogenase (LDH) into the cell culture supernatant from damaged cells was measured. The bar graph indicates the mean ± standard error of the mean (SEM) ( n = 2). * p 0.05, significant differences between control and each treatment group. # p 0.05, significant differences between prp (106–126)-treated cells in normoxia and hypoxia.

Fig. 2

Hypoxia regulates p65, p53, and p21 protein expression. (A) SH-SY5Y cells were incubated with 200 μm prion protein fragment, prp (106–126), for 24 hours following growth for 12 hours in normoxic conditions (21% oxygen tension) or hypoxic conditions (1% oxygen tension). Treated cells were immunostained with HIF-1α antibody (green) and fluorescence was examined. (B) HIF-1α protein levels in SH-SY5Y cells treated as described in (A).Brain anoxia expression levels were determined by western blot analysis and densitometric values are shown below the blot. The bar graph indicates the mean ± standard error of the mean (SEM) ( n = 2). * p 0.05, significant differences between control and each treatment group. (C) representative images of p65 expression in SH-SY5Y cells treated as described in (A). (D) SH-SY5Y cells were exposed to the indicated dose (μm) of prp (106–126) in normoxia or hypoxia, and then immunostained with p53 antibody (green) and observed for fluorescence. (E) western blot analysis of p65, p53, and p21 expression in SH-SY5Y cells treated as described in (A).

Fig. 3

HIF-1α is involved in the neuroprotective effect of hypoxia. (A) SH-SY5Y cells were pretreated with 2 μm deferoxamine (DEF) under normoxia for 12 hours before treatment with 200 μm of prion protein fragment, prp (106–126), for 24 hours.Brain anoxia treated cells were photographed with a light microscope (×200). Cell viability was measured by the crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control is presented. The bar graph indicates the mean ± standard error of the mean (SEM) ( n = 2). * p 0.05, significant differences between control and each treatment group; # p 0.05, significant differences between cells pretreated with and without 2 μm DEF before treatment with prp (106–126). (B) SH-SY5Y cells were pretreated with 0.04 μm doxorubicin (DOX) under hypoxia for 12 hours before treatment with 200 μm of prp (106–126) for 24 hours. Treated cells were photographed with a light microscope (×200).Brain anoxia cell viability was measured as described in (A). The bar graph indicates the mean ± SEM ( n = 2). * p 0.05, significant differences between control and each treatment group; # p 0.05, significant differences between cells pretreated with and without 0.04 μm DOX before treatment with prp (106–126). (C) western blot analysis of HIF-1α, p65, p53, and prp C from SH-SY5Y cells treated with 2 μm DEF under normoxia or with 0.04 μm DOX under hypoxia for 24 hours. Β-actin was used as a loading control.

Fig. 4

Lentiviral short hairpin (sh) RNA knockdown of hypoxia-inducible factor-1 alpha (HIF-1α) sensitizes SH-SY5Y cells to prion protein fragment [prp (106–126)]-induced neuronal cell death. (A–D) HIF-1α-shrna or mock transfected SH-SY5Y cells were cultured in 21% or 1% oxygen tension for 12 hours, and then treated with the indicated dose (μm) of prp (106–126) for 24 hours.Brain anoxia treated cells were photographed with a light microscope (×200). Cell viability was measured by the crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control is presented. The bar graph indicates the mean ± standard error of the mean (SEM) ( n = 2). * p 0.05, significant differences between control and each treatment group; # p 0.05, significant differences between the cells transfected with HIF-1α-shrna and with mock transfection. (E) transcription level of HIF-1α was determined in HIF-1α-shrna or mock transfected SH-SY5Y cells incubated in 21% or 1% oxygen tension for 24 hours. Total RNA was extracted and reverse transcription-polymerase chain reaction (RT-PCR) was performed.Brain anoxia reverse transcription-polymerase chain reaction products were separated on 1.2% agarose gel and stained with ethidium bromide. Β-actin was used as an internal control. Marker: 100 base pair DNA ladder. (F) representative immunoblots showing HIF-1α, p65, p53, and cellular prion protein (prp C) levels in SH-SY5Y cells treated as described in (E). Β-actin was used as a loading control.

Fig. 5

Cellular prion protein (prp C) knockout affects the hypoxia-mediated neuroprotective effect against prion protein fragment, prp (106–126). (A) ZW 13-2 and zpl 3–4 cells were incubated with 300 μm prp (106–126) under normoxic or hypoxic conditions. In brief, ZW 13–2 and zpl 3–4 cells were cultured in 21% or 1% oxygen tension for 12 hours, and then treated with 300 μm prp (106–126) for 24 hours.Brain anoxia treated cells were photographed with a light microscope (×200). (B) cell viability was measured by the crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control is presented. The bar graph indicates the mean ± standard error of the mean (SEM) ( n = 3). * p 0.05, ** p 0.01, significant differences between control and each treatment group; # p 0.05, significant differences between ZW 13–2 and zpl 3–4 treated with prp (106–126) under hypoxia. (C) AD- prnp or AD-empty transfected zpl 3–4 cells were incubated with or without 300 μm prp (106–126) for 24 hours. Cell viability was measured by annexin V assay. M1 represents the population of annexin V-positive cells.Brain anoxia bar graph indicates the averages of annexin V-positive cells. ** p 0.01, significant differences between control and each treatment group. (D) cells were treated with 300 μm prp (106–126) for 24 hours after exposure to the viruses, and release of lactate dehydrogenase (LDH) into the cell culture supernatant was measured. Bar graph indicates the mean ± SEM ( n = 3). ** p 0.01, significant differences between control and each treatment group. (E) hypoxia-inducible factor-1 alpha (HIF-1α) and prp C protein levels in ZW 13–2 and zpl 3–4 cells exposed to 300 μm prp (106–126) under normoxia or hypoxia for 24 hours. (F) representative immunoblots showing prp C protein levels in the AD- prnp or AD-empty transfected zpl 3–4 cells treated as described in (C). (G) western blot analysis of p65 and phospho-ERK expression in ZW 13–2 and zpl 3–4 cells treated as described in (E).Brain anoxia densitometric values are shown below the blot. Β-actin was used as a loading control.

The human prion protein fragment, prp (106–126), may contain a majority of the pathological features associated with the infectious scrapie isoform of prp, known as prp sc. Based on our previous findings that hypoxia protects neuronal cells from prp (106–126)-induced apoptosis and increases cellular prion protein (prp C) expression, we hypothesized that hypoxia-related genes, including hypoxia-inducible factor-1 alpha (HIF-1α), may regulate prp C expression and that these genes may be involved in prion-related neurodegenerative diseases. Hypoxic conditions are known to elicit cellular responses designed to improve cell survival through adaptive processes.Brain anoxia under normoxic conditions, a deferoxamine-mediated elevation of HIF-1α produced the same effect as hypoxia-inhibited neuron cell death. However, under hypoxic conditions, doxorubicin-suppressed HIF-1α attenuated the inhibitory effect on neuron cell death mediated by prp (106–126). Knock-down of HIF-1α using lentiviral short hairpin (sh) RNA-induced downregulation of prp C mrna and protein expression under hypoxic conditions, and sensitized neuron cells to prion peptide-mediated cell death even in hypoxic conditions. In prp C knockout hippocampal neuron cells, hypoxia increased the HIF-1α protein but the cells did not display the inhibitory effect of prion peptide-induced neuron cell death. Adenoviruses expressing the full length prnp gene (ad- prnp) were utilized for overexpression of the prnp gene in prp C knockout hippocampal neuron cells.Brain anoxia adenoviral transfection of prp C knockout cells with prnp resulted in the inhibition of prion peptide-mediated cell death in these cells. This is the first report demonstrating that expression of normal prp C is regulated by HIF-1α, and prp C overexpression induced by hypoxia plays a pivotal role in hypoxic inhibition of prion peptide-induced neuron cell death. These results suggest that hypoxia-related genes, including HIF-1α, may be involved in the pathogenesis of prion-related diseases and as such may be a therapeutic target for prion-related neurodegenerative diseases.