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From the National Creutzfeldt-Jakob Disease Surveillance Unit* and Pathology (Neuropathology), School of Molecular and Clinical Medicine, University of Edinburgh, Edinburgh; the Biological Gerontology Group, School of Medicine and School of Biological Sciences, University of Manchester, Manchester; and the Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, United Kingdom
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Abstract |
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Studies of mice ablated for the prion protein gene (PrP0/0), have shown it to be nonessential for viability.9 PrPC has been implicated in cell-cell signaling, cell adhesion, and other cell-specific roles, for example, during embryogenesis.10-12 Others have suggested that it may have a specific neuronal role, supported by its presynaptic location in axonal terminals,13 for example, in regulation of circadian rhythm14,15 or GABAA-receptor-mediated fast inhibition.16 More recently PrPC has been proposed to have a role in the cellular defense against oxidative stress. Pheochromocytoma cells selected for resistance to oxidative stress express higher levels of PrPC than wild-type cells.17 Moreover, tumor prostate spheroids subjected to increasing oxidative stress display increased PrPC expression,18 as do NT-2 cells exposed to heat shock,19 and cerebellar cells from PrP0/0 mice are more susceptible to oxidative stress than are wild-type cells,20,21 and studies with PrP0/0 mice have shown increased levels of oxidative stress markers in vivo compared to wild-type animals.22-24 Additionally, it has been suggested that the pathogenesis of prion diseases could in part be because of a loss of PrPC function in the management of oxidative stress.25-27
When hypoxic damage occurs, oxidative stress is a potentially important cause of cellular injury and death. In mounting protective responses, cells may differentially express a number of proteins, including antioxidants, which limit the damage and initiate repair processes.28,29 If PrPC does have an antioxidant role, it might be induced in response to oxidative stress in neurons. To examine our hypothesis we chose cerebral ischemia (CI) and perinatal hypoxic-ischemic injury (HII) as common human disorders exhibiting oxidative stress responses in the central nervous system, as well as a mouse model of CI, and used immunohistochemical and in situ hybridization analyses to compare PrPC expression in hypoxic and nonhypoxic brain tissue sections.
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Materials and Methods |
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Autopsy records were used to identify cases in which clinical history and postmortem neuropathological examination indicated that recent cerebral infarction had occurred and directly contributed toward death. Formalin-fixed, paraffin-embedded tissues from 12 such cases were obtained from the Department of Pathology (Neuropathology), School of Clinical and Molecular Medicine, University of Edinburgh, Edinburgh. Samples from these cases were processed at postmortem with a fixation regime of at least 2 weeks in 10% neutral formalin followed by paraffin embedding. The range of ages of the cases was 42 to 90 years, postmortem intervals were 24 to 72 hours, and the delay between onset of symptoms to death was <24 hours to 40 days. Details of the cases are summarized in Table 1
. The brain regions studied varied between cases depending on the site of infarction and included both white and gray matter regions. Three tissue blocks containing infarcts were available for case number A1 whereas single blocks represented the rest of the cases. The perinatal HII series consisted of five cases in which cerebral hypoxia was diagnosed on postmortem microscopic examination, and brain tissues from three perinatal cases that did not display neuropathological injury were used as negative controls. Hypoxic cases are summarized in Table 2
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CI in Mice
All mice used in this study were a kind gift from J. Manson (Institute for Animal Health, Edinburgh, UK). The role of PrP in ischemic brain damage was investigated in adult male wild-type 129/Ola (PrP+/+) mice, inbred PrP-null mice 129/Ola (PrP0/0), and the heterozygous cross. Focal CI was induced by electrocoagulation of the left middle cerebral artery (MCA)30 and mice were studied 24 hours later. This procedure, with the electrocautery consistently performed at the same site, produces a highly reproducible pattern of brain damage with little, or no, mortality. Body temperature was maintained at 37°C throughout surgery and the postoperative recovery. Twenty-four hours later, the mice were reanesthetized and trans-cardiac perfusion performed using either periodate-lysine-paraformaldehyde (final concentration of 2% formaldehyde, pH 7.4, for immunohistochemistry) or with 4% phosphate-buffered formaldehyde (for in situ hybridization). In addition, the brains of further mice were either immersion-fixed in formol-saline for in situ hybridization studies or were removed fresh for the determination of the infarct size.
Immunohistochemistry
For immunohistochemistry analysis 5-µm sections were cut from fixed brain tissue blocks. Sections were mounted on glass slides (Superfrost Plus; BDH, Poole, UK), dewaxed, rehydrated, and endogenous peroxidases quenched by treatment with 0.3% hydrogen peroxide in methanol for 30 minutes. The primary antibodies, dilutions, incubation conditions, and antigen retrieval methods used in this study are listed in Table 3
. Sections from human adult and pediatric brain were treated with both the anti-ßAPP antibody MAB348 (Chemicon Int., Temecula, CA) and the anti-PrP monoclonal antibody 3F4 (DAKO, Glostrup, Denmark), diluted in 20% rabbit serum in Tris-buffered saline (20 mmol/L Tris-HCl (pH 7.6), 137 mmol/L NaCl). After primary antibody incubation and washing in Tris-buffered saline, sections were incubated with a biotinylated rabbit anti-mouse antibody (Scottish Antibody Production Unit, Edinburgh, UK) diluted 1:200 in 20% rabbit serum in Tris-buffered saline for 30 minutes. The staining pattern was visualized using the tyramide signal amplification methodology (CSA, DAKO). Sections were counterstained with hematoxylin before mounting in DPX (BDH).
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In Situ Hybridization
Tissue sections from both the human perinatal HII case (P3) and murine CI models (n = 3) were subjected to in situ hybridization analysis using the method described previously.3 In summary tissue sections were rehydrated, partially digested with proteinase K, and hybridized with human or mouse-specific PrP cRNA probes labeled with digoxigenin (Roche Diagnostics, Lewes, UK). Unduplexed RNA molecules were removed using RnaseA in high salt, followed by washing with increasing stringency. Hybridized sections were treated with anti-digoxigenin Fab fragments labeled with alkaline phosphatase (Roche Diagnostics) and visualized by using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as the chromagen. cRNA molecules complimentary to the test probes served as controls. The developed tissue sections were analyzed and images captured as above. Other sections were stained with 0.1% toluidine blue or 0.1% cresyl violet to show the morphological appearance of the ischemic lesion.
Ischemic Lesion Size Measurement
The ischemic lesion size was assessed in a total of 19 129/Ola (PrP+/+) mice, 17 129/Ola (PrP0/0) mice, and 6 heterozygous crosses of these, in more than four experiments throughout a 2-year period. The animals were anesthetized with halothane, killed by decapitation, and their brains removed. All brains were coded before sections were cut to ensure that measurements were made blind. Coronal sections, 500 µm thick, were cut using a 735M tissue Vibroslice, and the slices immediately immersed in 0.1% tetrazolium blue chloride (containing 1.25% dimethyl sulfoxide and 2 mmol/L MgCl2 in 0.1 mol/L phosphate buffer, pH 7.4). Slices were incubated for 30 minutes at 37°C after which the infarct was visible as an unstained region of the section. The sections were then fixed in 4% phosphate-buffered formaldehyde (pH 7.4), for a minimum of 24 hours at 0 to 4°C before image analysis. An indirect method was used to calculate infarct volume using a semiautomated image analysis system (Imagan 2; Leitz/Kompira, UK). Three measurements were recorded: 1) a, area of the infarct, mm2; 2) b, area of infarcted left hemisphere, mm2; 3) c, area of the nonischemic right hemisphere, mm2. We observed enlargement of the brain hemisphere on the side that had undergone MCA occlusion. To compensate for this increase in the size of the hemisphere the proportional increase in the size of the infarcted lobe was calculated as follows: Proportional increase because of swelling of the hemisphere (d) = (b – c)/(b + c); infarct area (mm2) corrected for swelling (e) = a/(1 + d); giving the total infarct volume = 
(e).t (where t is section thickness). The data on infarct size were then calculated as the mean values and SD for each of the three PrP genotypes. An analysis of variance was used to compare the data from successive experiments and significant differences between the groups identified using the Scheffé multiple comparison test.
Vascular Structure Determination—Perfusion with Latex and Indian Ink
Mice were prepared for surgery as described above. The heart was exposed and the animal perfused with heparinized saline until the draining fluids were cleared of red blood cells (usually 1 to 2 ml of saline). The animal was then perfused with a latex solution (Neoprene latex 671A; Omya, UK) containing 10% waterproof Indian ink. The animal was decapitated and the brain carefully dissected from the skull using fine dissecting scissors and fixed by immersion in 4% phosphate-buffered paraformaldehyde for visual analysis. Four mice of each genotype were used for the analysis
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Results |
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The adult CI cases examined in this series displayed areas of focal infarction involving both gray and white matter, with typical vascular lesions circumscribed by PrP immunoreactivity within swollen axonal bulbs together with a punctate background staining pattern (Figure 1)
. This PrP-penumbral-staining phenomenon was demonstrated within serial sections of the same hypoxic foci. Overall 10 of the 12 cases showed infarct-associated PrP immunoreactivity although the staining intensity varied between cases and heterogeneous patterns of PrP deposition were seen (cases A2 and A9 were PrP-negative). Of these 10 cases all showed a high degree of punctate PrP staining (Figure 1, f to i
, with h as a control), whereas 5 of the cases showed large amounts of axonal staining (cases A1, A3, A4, A7, and A12) (Figure 1e)
, although positive axons were seen in all cases.
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. Serial sections demonstrated that ßAPP immunoreactivity was invariably present in axons immunoreactive for prion protein. In cases with gray matter lesions, similar patterns of PrP deposition were seen (Figure 1j)
, and in addition PrP-positive neuronal soma (morphologically identified) were detected. Here immunopositivity was found to exist in spatially distinct clusters and undamaged adjacent brain areas did not show such PrP-expressing cells (Figure 1k)
. Control tissues and nondamaged regions from the test cases did not show any such positive staining for PrP or ßAPP. The length of the postmortem interval did not seem to correlate with the degree of PrP staining with both short (24 hours) and long (72 hours) intervals resulting in varying degrees of staining. The same was true for the disease duration with both long and short durations resulting in variable degrees of staining. The two cases that showed no infarct-associated PrP staining (A2 and A9) differed in disease duration (A2 < 24 hours, A9 = 28 days). Other cases with similar disease time frames (cases A7 and A10, short duration; A4, A8, and A11, longer duration) were strongly positive for PrP.
Perinatal HII Cases
In the perinatal cases, hypoxic regions were identified by the presence of eosinophilic neurons and reactive microglia. All cases studied showed PrP staining associated specifically with the hypoxic regions. The majority of immunostained cells occurred in clusters and were morphologically identified as neurons (Figure 2, a and b)
. Prion protein immunostaining was not demonstrated in nonhypoxic control tissue, nor was it present in undamaged regions within the hypoxic cases. The pediatric cases differed from the adult ones in that the PrP staining was primarily within the neuronal soma rather than axonal.
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. No cell types other than neurons were found to be expressing PrP mRNA at detectable levels. Murine Stroke Model
In this study, permanent MCA occlusion caused reproducible damage in each strain of mouse but no major neurological deficits, or mortality, were observed throughout the 24-hour period after the onset of ischemia. The size, severity, and pattern of lesion was consistent within each PrP genotype of mouse of mice studied (Table 4)
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Examination of toluidine blue-stained sections showed ischemic brain damage in the PrP+/+, PrP+/0, and the PrP0/0 mice (serial coronal sections shown in Figure 3
). The infarct contained no recognizable neurons but many vacuolated cell profiles with a small, dark, pyknotic nucleus; an appearance typical of cells that have undergone ischemic cell death.31
The only surviving neurons were found at the edges of the lesion. Extensive neuronal loss was seen in the striatum of the PrP-null mice, but not in the wild-type mice or the PrP+/0 heterozygous cross.
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. The lesion in 129/Ola PrP+/0 heterozygous mice was intermediate between those of the null and wild-type mice. Further studies revealed that the vascular structures in PrP+/+, PrP+/0, and PrP0/0 mice was similar suggesting that the infarct volume depends on PrP expression rather than being mediated by a developmental difference in cerebral vasculature between the three lines (Figure 4)
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50% of the difference between the lesion size between null and wild-type mice (Table 4)
. LSD (least significant difference, post hoc multiple comparison) showed significant differences between each of the three groups of mice. PrP Immunocytochemistry and in Situ Hybridization
In nonoperated mice, and in the contralateral hemisphere of mice with the MCA occlusion, PrPc was detected as a faint punctate staining of occasional neuronal perikarya, as reported previously.32
In contrast, within the penumbra of the infarct, where the neurons are sublethally damaged, many neurons showed PrP immunoreactivity with both PrP antibodies used. In addition, PrP immunoreactivity was associated with neuronal cell bodies and as diffuse, punctate staining of the neuropil in the ischemic hemisphere (Figure 5a)
. Similar increases in PrP immunoreactivity have also been observed in response to MCA occlusion in C57BL mice (Williams A, Davies I, Fotheringham A; data not presented) and in normal rats (Horsburgh K, Wellcome Surgical Institute, Glasgow; and Williams A, data not shown). No PrP immunoreactivity was present in the core of the infarct and no increases in PrP staining were seen in the contralateral side of the brain in ischemic (Figure 5b)
or nonoperated mice. The use of normal rabbit sera and antibodies raised against irrelevant antigens failed to show any staining indicating the specificity of the anti-PrP antibodies.
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. No mRNA signal was detected in the 129/Ola PrP0/0 mice at the longer or shorter exposure times. |
Discussion |
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In the CI cases PrPC accumulation was observed in all positive cases as a punctate background of immunopositivity and in addition in half the cases much of the PrP is seen within damaged axons in the brain, frequently coinciding with ßAPP immunopositivity. The ßAPP protein is known to be up-regulated during CI and to accumulate within damaged axons as a result of disrupted anterograde axonal transport.34,35 Thus, PrPC expression within hypoxic lesions might simply represent a passive result of deranged neuronal protein trafficking rather than an induced response to injury. However, because neuronal somata also displayed PrPC immunostaining, it is possible that PrPC synthesis is increased after hypoxia, even if in some cases cytoskeletal damage is contributing physically toward the axonal accumulation. Thus, it is likely that the axonal co-localization of ßAPP and PrPC are independent consequences of the ischemic injury rather than demonstrative of a functional relationship. It is interesting to note that in some (but not all) cases of Alzheimer’s disease PrPC and the amyloid ß fragment co-localize in amyloid plaques,36 although the significance of this observation is unclear at present.
In our study 10 of the 12 cases of adult CI showed immunopositivity for PrP. The two negative cases were not similar in that the disease durations were very different (<24 hours versus 28 days). There are many potential variables in the human samples used here such as time variables, sample age, degree of fixation, and sample handling, which could contribute to the variations seen in all of the cases, however it seems most likely that the location of the ischemic lesion plays a major role in determining the resulting cellular responses and therefore it could be argued that these cases are not directly comparable. Nevertheless our finding that PrP accumulation is seen in almost all of these diverse cases is most striking.
In situ hybridization analysis of a perinatal HII case demonstrated an increased PrP mRNA signal in those regions containing PrPC immunostaining neurons. We propose that the most likely mechanism to account for the observed neuronal immunoreactivity of PrPC accumulation is a transcriptional up-regulation of prion protein gene expression. This suggestion is backed up by research from Shyu and colleagues,37 although a decrease in mRNA turnover in these cells cannot be excluded. The transcriptional stimuli regulating PrPC expression are unknown, although inflammatory cytokines and reactive oxygen species, both of which induce PrPC in vitro18,38 are produced within the penumbra of ischemic lesions. Further analysis of this phenomenon and the utilization of cell culture and animal models will allow us to address these questions more directly.
The studies of experimental ischemia in mice (permanent middle artery occlusion) confirmed the results obtained on human tissue. In mice, increased PrP mRNA and PrP immunoreactivity were seen associated with neuronal soma in the penumbra of the ischemic lesion. The difference in cellular location between the mouse model and human material could reflect the fact that the lesion in the mice was more acute and affected a greater relative proportion of the brain so that the increased PrPC was detected in neuronal cell bodies only, and not in axons after subsequent transport down neuronal processes.
The functional importance of PrPC, with a protective role after neuronal injury, was further emphasized in studies using PrP-null mice. All of the mice used in this study had an inbred 129/Ola background39 allowing us to make direct and accurate comparisons of the lesion size in wild-type 129/Ola (PrP+/+) and PrP-null (129/Ola PrP0/0) mice without confounding factors because of diverse genetic backgrounds. The lesion size in the 129/Ola PrP+/+ and in the 129/Ola PrP0/0 mice was highly reproducible, with only a small range in lesion size within the genotypes (but differing volumes between genotypes). These differences could not be attributed to variations in vascular structures because it was shown that these structures are similar in PrP+/+, PrP+/0, and PrP0/0 mice. The lesion was significantly larger in PrP0/0 mice compared to mice of the same strain but possessing either one or two copies of the PrP gene. This increase in lesion size in the PrP-null mice would support the hypothesis that some neuronal populations sublethally damaged in mice containing a functional PrP gene, but able to survive, were not able to do so when devoid of PrP. Reintroduction of a single PrP allele, as in the hemizygous mice, was only partially protective suggesting a quantitative effect in which the level of PrPC is limiting, further reinforcing a potential neuroprotective role for the protein.
Injured neurons increase synthesis of protective proteins and decrease production of nonessential molecules,40 and so our data supports the hypothesis that PrPC may have a neuroprotective role. Although other reports suggest a role for PrP in the oxidative stress response, it is not possible from these results to dissect out a distinct biochemical function for PrPC in the hypoxically injured brain. Understanding the physiological role of PrPC may help to elucidate the pathological processes associated with the prion diseases, and consequently suggest treatment strategies. PrPSc, the disease-associated isoform of prion protein, may have an aberrant neuroprotective function resulting from alteration in its secondary structure compared to PrPC. Indeed, prion infected cells have been shown to exhibit increased susceptibility to oxidative damage.24,41,42 Oxidative stress has also been implicated in the pathogenesis of a number of human neurodegenerative conditions, such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis43,44 and so PrPC may also play a part in these conditions. Further analysis of PrPC expression and function in normal and diseased brain tissues, in cell culture and in animal models of hypoxia and other neurological conditions may provide further understanding of the role of PrP in this and other neurological conditions.
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Acknowledgements |
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Footnotes |
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Supported by the Medical Research Council (G9627376), the European Union (EC Biotech 4-98-6064), the University of Edinburgh (Faculty of Medicine Vacation grant to P.B.), and the Welcome Trust (Vacation Research scholarship to A.M.).
Current address of A.W.: Royal Veterinary College, London, UK.
Accepted for publication March 30, 2004.
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