Prion Disease Progressive Neurodegenerative Disorders In Humans Animals Biology Essay

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Prion disease or transmissible spongiform encephalopathies (TSEs) are a group of progressive neurodegenerative disorders that affects both humans and animals due to its causative agent; the prion protein. (Linden et al., 2008). The term 'prion' is used to describe the 'proteinaceous' and 'infectious' nature of this specific protein (Prusiner, 1982) whose abnormal scrapie isoform, PrPsc is said to be the main constituent involved in the progression of this disease (Prusiner, 1998). Causing bovine spongiform encephalopathies (also known as 'mad cow disease') in cattle and a degenerative neurological disorder in humans known as Creutzfeldt-Jakob disease (CJD) in humans, prion diseases are incurable and invariably fatal (Linden et al., 2008). The production of the disease-inducing isoform PrPsc, relies on its conversion from PrPc, the normal prion protein isoform (Pan et al., 1993) which is described by Martins et al., (2001), to be a 'glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety'. Although the physiological properties and function of PrPc have not fully been identified (Mitsios et al., 2007), new lines of research have suggested its role in mounting neuroprotective cellular responses. Proposed to have a role in defending cells against oxidative stress after ischemic insult (e.g. stroke or hypoxia), PrPc's neuroprotective effect on injured neural tissues could provide a means of finding a therapeutic approach to treat such conditions (McLennan et al., 2004). Although other studies detail its over-expression in protein synthesis, stress protective signalling and gastric cancer cell lines, the molecular mechanism by which PrPc works, is still unclear. This review presents evidence that details the role of PrPc in neuroprotection as well as the prospective and potential strategies that hope to further our understanding of this protein, which would prove essential in suggesting strategies of treatment.


The discovery of PrP and its molecular marker, PrPsc, transformed research on scrapie (sc) and other related diseases. Truncated PrPsc, known as PrP 27-30 (due to its molecular weight of 27kD- 30kD), is resistant to proteolysis and led to the identification of Prnp, a gene that encodes the prion protein, PrPc (Bolton et al., 1984). Expressed in the nervous system but in varying amounts, the prion protein is found primarily among neurons, but also has been found to be in bone marrow, various parts of the immune system as well as in blood and peripheral tissues (Linden et al., 2008).

The PrPc encoder gene, Prnp, is located on chromosome 2 in mice (Prnp) and chromosome 20 in humans (PRNP) (Basler et al., 1986). Both isoforms of the protein consist of 208 - 209 amino acids (aa's), with the Prnp genes open reading frame (ORF) being located entirely on one axon (Basler et al, 1986). This is shown in Figure 1.

Fig. 1 - Diagram detailing the Prnp gene in Syrian hamsters. (Taken and modified from Prusiner, 2004) the Prnp gene encodes a protein of 254 amino acids (green) and cleaves 22/23 amino acids from the amino and carboxyl termini. This primary translation product (green) shows PrPc's octarepeats, glycosylphosphatidylinositol (GPI) anchor and disulphide bond (represented by 's-s'). Once cleavage and maturation have occurred both PrPc (brown) and PrPsc (orange) consist of 209 amino acids. Due to protease resistance, PrPsc (cream) is truncated and forms PrP 27-30 (Prusiner,2004).

Once the primary translation product has been cleaved (via the removal of 22 aa's), the remaining 209 aa, PrPc, can then be exported to the cell surface as an N-glycosylated glycosylphosphatidylinositol (GPI) anchored residue. Its main features (Fig.2) consist of a flexible amino (NH2) terminal containing octapeptide repeats, a globular domain containing three α helices with an interspersed β pleated sheet and a single disulphide bond (Reik et al.,1996).

Copper, implicated in providing neuroprotection (discussed later on), binds to the octapeptide region as well as histidine 96 and 111 on the flexible tail and also has a role in stimulating endocytosis of PrPc (Prusiner, 2004).

Fig. 2 - Diagram showing the structure of the mammalian prion protein, PrPc. (Taken from Pushie & Vogel, 2007) The N domain consisting of a flexible tail and octarepeats provide sites for copper binding, as do the histidine sites. The C domain shows the three α helices and a β sheet.

Fig.3 - Structures of PrPc and PrPsc. (Taken from Prion Biology Laboratory, 2010). This diagram shows PrPc (A) which is dominated by α helices and only one β sheet, and PrPsc (B), which has higher β sheet content than α helix content.PrPc differs from its disease-inducing isoform in their structures. Whilst PrPc is an α-helical rich protein, PrPsc is a compact β sheet structure (Fig.3), which explains its resistance to proteolysis (Pan et al., 1993).

They do however share the same amino acid sequence as well as the same single copy encoding gene, Prnp, so the ability to distinguish between the two isoforms is essential (Riek et al., 1986). Although it has been determined that the properties of each isoform are due to post translational modifications, the conformational conversion and modification of PrPc to PrPsc has been a widely researched area (Linden et al., 2008).

The gain of function hypothesis provides an explanation for this conversion, attributing PrPsc as an 'anomalous conformer' of the normal prion protein isoform, PrPc. The loss of function hypothesis however details the fact that prion disease is caused by a loss of PrPc (Linden et al., 2008), this theory in regards to neuroprotection will also be further discussed.


Initial studies conducted, with the hope of exposing PrPc's physiological function and role were undertaken. One such study conducted on mice with prion protein ablation showed that the protein had no effect on viability as its absence elicited normal behaviour and development in mice (Bueler et al,. 1992). The knockout mice (Prnpo/o) were generated by Bueler, et al. (1992) by disrupting the Prnp allele of murine embryonic stem (ES) cells. This strategy involved homologous recombination whereby PrP codons 4-187 of the 254 codon open reading frame (ORF) were replaced by a neomycin phosphotransferase (neo) gene. Not only did the mice homozygous for the disrupted gene (Prnpo/o) express truncated PrP mRNA and no detectable PrP fragments, approximately a quarter of the offspring of mated hemizygous mice generated homozygous knockout mice, illustrating normal behaviour and reproductive qualities. More recently, knockout studies have been conducted since the emergence of several physiological roles of PrPc in cell adhesion, signalling and neuroprotection (Reilly, 2000).

Many areas of PrPc's role in neuroprotection were studied by McLennan, et al. (2004) in relation to hypoxic brain damage. One area focussed on PrPc's role in cerebral ischemic (CI) brain damage by looking at knockouts from a murine stroke model. Electrocoagulation of the left middle cerebral artery (MCA) at the same site in each mouse strain caused CI in 19 adult male wild type 129/Ola19 (PrP+/+) mice, 17 inbred 129/Ola19 (PrPo/o) PrP-null mice and 6 heterozygous crosses of these 129/Ola19 (PrP+/o). After anaesthetisation and removal, brain sections were stained to reveal the infarct size of each mouse strain. The infarct, described in Mosebys dictionary of complementary and alternative medicine as being the 'localised tissue death resulting from an interruption of blood supply to that area' contained cells that were reminiscent of those who had suffered ischemic cell death and the only identifiable neurons were found at the lesion parameters. Both the wildtype and heterozygous cross show no neuronal loss whilst the PrP null mice did, demonstrating a potential neuroprotective role for the protein. Unlike the wildtype which showed no lesions, the PrP null mice exhibited cortical damage whose lesion reached as far as the striatum extending both dorsally and ventrally, whilst the lesion in the heterozygous cross lay in between. The significant larger lesions in the PrPo/o mice in comparison to the wildtype or heterozygous cross illustrates that some neurons aren't able to withstand death when lacking PrP. Partial protection from neuronal death by possessing one copy of the PrPgene, perhaps shows that the level of PrPc can quantifiably affect its neuroprotective capacity. Each strain of mice had the same 129/Ola background and the vascular structures of each were examined and shown to be of similar profiles. This ensured that the lesion size was due to PrP expression and not another variable supporting the proposed role of PrPc in neuroprotection (McLennan et al., 2004).


Other studies with knockout mice have provided a means of examining PrPc in relation to oxidative stress. Oxidative damage occurs due to the production of reactive oxygen species (ROS) (Davies, 1995), and PrPc, which has been suggested to possess and antioxidant role, can limit the damage caused by oxidative stress in neurons (McLennan et al., 2004)

Lipid peroxidation and protein oxidation are both indicators of oxidative stress. The more oxidative damage occurs, the more lipid peroxidation will produce reactive aldehydes, and the more protein oxidation will produce carbonyls (Halliwell & Gutteridge, 1985). The levels of two aldehydes measured by Wong, et al. (2001) that are indicative of lipid peroxidation are malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). In order to ensure peroxidation of non-specific aldehydes didn't impair results, butylate hydroxytoulene (BHT), a chain breaking antioxidant was administered. By comparing the levels in the brains of PrP knockout mice (Prnp-/-) with age-matched wildtype mice of the same genetic background, it was seen that increased levels of MDA and 4-HNE were found in the former. The level of carbonyl compounds produced by protein oxidation was also higher (~ 49%) in Prnp-/- than in the wildtype. A correlation between loss of PrP and an increased level of oxidative stress markers (MDA, 4-HNE and carbonyl compounds) in the brains of Prnp-/- mice can be seen from this report, however, care must be taken to ensure that the extent of oxidative stress isn't due to other cellular repair mechanisms compensating for the damage (e.g. damaged proteins undergoing ubiquination by proteolysis) (Wong et al., 2001).

Other reports correlating PrPc and oxidative stress have further developed our understanding of this prion protein, particularly its relation to brain injury such as hypoxia (McLennan et al., 2004). Described by the National Institute of Neurological Disorders and Stroke (2010) as a 'condition in which there is decreased oxygen supply to the brain', hypoxia, due to oxygen deprivation, causes cellular injury and depending of the extent of hypoxic damage, death (McLennan et al., 2004). McLennan et al (2004) used perinatal hypoxic- ischemic injury (HII) and human and mouse models of cerebral ischemia (CI) as disorders reminiscent of oxidative stress to investigate the role of PrPc. In conjunction with this, both PrPc expression in hypoxic and non hypoxic brain tissue were examined via immunohistochemical and in situ hybridisation. Initial western blot studies revealed that the characteristics of PrP expressed in hypoxic brain tissue weren't due to the scrapie isoform PrPsc. This, combined with studies (Esiri et al., 2000) revealing the possible upregulation of cellular PrP in the human brain after injury, promoted McLennan et al (2004) to propose their observed immunostained PrP as a reflection of the presence of PrPc. Their findings include the presence of PrPc immunoreactivity within white and gray matter damage in human CI. Ten out of the twelve cases studied showed PrP immunopositivity whilst the other two negative cases didn't. This difference however could be accounted for due to its varying disease duration (<24hrs vs 28 days), perhaps suggesting that the size of the ischemic lesion contributes to the cellular response. PrP accumulation seen in all the other cases, reinforce PrPc's involvement in response to hypoxic damage. An issue to note was that although all the positive CI cases showed a high degree of punctate staining with over half of which also showed staining in the damaged axons of the brain, βAPP staining (β amyloid precursor protein) also coincided with this. Known to accumulate as a result of damaged axonal transport and in response to CI, βAPP's immunopositivity may reflect a passive expression of PrPc due to poor axonal transport. If this factor did somewhat contribute to PrPc accumulation, the fact that the positive control also showed an increase in PrPc immunostaining after hypoxic damage it was suggested that the axonal build up of PrPc and βAPP are independent occurrences rather than a result of fundamental relational between the two (McLennan et al., 2004). The same study conducted on a mouse model from the same authors confirmed the human CI PrPc results. Increased PrP mRNA and immunostaining were seen in the lesion areas, supporting neuronal PrPc having an involvement to hypoxic events.

In situ hybridisation studies of a perinatal HII case showed increased PrP mRNA signals in the areas expressing PrPc immunostaining (McLennan et al., 2004). The suggestion that PrPc accumulation is due to transcriptional upregulation of the prion protein and the induction of cytokines and ROS need to be further analysed.

In supporting the expression of PrPc in brain injury, Mitsios et al (2007) reported its effects in the plasma and peri-infarcted tissue after stroke. PrPc was shown to be upregulated by 1.5 - 5.3 fold, from western blotting studies in gray matter peri-infarcted tissue, and by 1.5 - 6.4 fold in infracted tissue. When oxygen-glucose deprivation (OGD) was induced, PrPc expression also increased in peri-infarcted positive, human foetal neurons (HFN). This expression could promote cell damage resistance associated with hypoxic ischaemic damage. The overall higher circulatory levels of PrPc in the plasma of patients with ischemic stroke compared to healthy adults (3.1 ng/ml vs 0.7 ng/ml), suggests that the modulation of PrPc expression may provide a good basis of developing potential therapeutic strategies and treatment of stroke.


The suggested role of PrPc in response to oxidative stress was further demonstrated by Qin et al (2007) who looked at the modulation of PrPc by ataxia-telangiectasia mutated (ATM) mediated transcription in respsonse to copper-induced oxidative stress. Not only did this provide information on copper Cu(II), but also the transcriptions effect of the prion by the upregulation of ATM. Oxidative stress in murine N2a (neuroblastoma) and HeLa cells, were induced by copper, whose high concentrations were expected to cause toxicity leading to ROS (reactive oxygen species) accumulation (Qin et al., 2007). Normal PrPc levels in N2a cells were compared with those in Cu(II) treated N2a cells, increasing 10.4 ± 3.2 fold once Cu(II)-treated, showing that Cu(II) triggers the rapid elevation of PrPc. In investigating whether this elevation occurs at the transcriptional level, RT-PCR (reverse transcription polymerase chain reaction) was carried out. The control β-actin mRNA levels were almost constant after exposure to copper whereas the Prnp mRNA levels increased from 1.53 ± 0.3 fold to 4.46 ± 0.7 fold. Higher levels of ROS accumulation, due to copper causing oxidative stress, was observed in PrP-knockout N2a cells than in the wildtype N2a cells, suggesting that elevated PrPc (induced by copper), could prevent ROS build-up. Through finding that PrPc binds at the transcriptional level, ATM mediated transcription involvement was tested. Phosphorylation and thus activation of ATM and its substrates p53 and MEK were observed, with MEK promoting ERK1/2 activation. The stimulation of these downstream stress-responsive molecules indicates ATM's involvement in copper- induced oxidative stress (Qin et al., 2007). These findings contribute to our understanding of PrPc's physiological function and it protective qualities against Cu(II)- induced oxidative stress.

PrPc, due to its superoxide dismutase (SOD) activity (an enzyme that is important for its antioxidant activity) (Brown et al., 1997), has been proposed to have an antioxidant role that contributes on alleviating cells from oxidative stress. In adjunct to copper, manganese (Mn) was another metal used as an inducer of oxidative stress by Choi et al (2007) who showed that PrP-null cells and PrPc cells induced Mn- cytotoxic cell death of ~ 59.9 and 117.6µm respectively. These protective qualities of PrPc were further examined by determining that the production of Mn- induced ROS was significantly reduced in PrPc cells compared to the knockout, perhaps elucidating PrPc's antioxidant properties (Choi et al., 2007). These findings, detailing the roles of copper and manganese in relation to PrPc and oxidative stress highlight the importance of conducting further studies to characterise their involvement with prion protein levels as well as the pathogenesis of prion diseases.


Studies demonstrating PrPc's role in neuroprotection after ischemic insult were furthered by studies that detailed PrPc as a modulator of stress protective pathways. Rambold et al (2008) showed the proteins dependency on the internal hydrophobic domain (HD) and GPI anchor, as well as the interconnected activity of toxic PrPsc and protective PrPc, providing insight into scrapie prions role in inducing apoptotic signalling in PrPc cells. In determining what PrPc structure conferred tolerance of stress, Rambold et al (2008) initially deleted the N terminal that has been known from previous studies on mice. This provided the means of identifying two novel domains; the HD and GPI anchor, as structures involved in PrPc's stress protective activity (as their deletion showed a loss in this activity). The mutant, PrP-CD4, containing a transmembrane domain and not a GPI anchor, showed no anti-apoptotic activity, supporting the need for PrP-GPI attachment to confer stress protective activity. In analysing the role of HD in mediating stress, the dimer formation, which is involved in stress signalling, was examined (Warwicker, 2000). It was revealed that endogenous PrPc in neuroblastoma (N2a) cells can form a dimer. PAGE and western blotting analysis showed a migration pattern similar to the wildtype PrP. The HD was shown to be a part of the dimer interface as PrP HD, a mutant impaired in dimer formation didn't provide any protection from stress induce cell death. Also scrapie infected cells (ScN2a), when exposed to heat or oxidative stress were shown to be more sensitive.

The mechanisms by which this signalling occurred is unknown but better understanding of PrPc and neurotoxic signalling of prion protein mutant would aid in developing strategies to treat and combat the pathogenesis of prion diseases. One particular study showed that hypoxia inhibits the mitochondrial apoptotic pathway (Seo et al, 2010). In the apoptotic pathway, mitochondria transmit cell death signals to the cytosol to trigger caspase production and ultimately death. Mitochondrial MTP factors and Bcl-2 expression is lost, but Seo et al (2010) show from western blot studies that hypoxia increased Bcl-2 protein expression, generates ROS and inhibits loss of MTP induced by PrP (106-126) - a neurotoxic prion fragment similar to PrPsc (Skulacher, 2000 ). Elevated levels of PrPc mRNA were found in neuroblastoma cells cultured under hypoxic conditions illustrating this proteins involvement in the protective mechanism of this condition. Although how it regulates neuroprotection is still undetermined, perhaps it contributes in inhibiting the mitochondrial apoptotic pathway.


The upregulation of PrP mRNA during hypoxia, provided as a basis for Liang et al (2007) to investigate the hypoxic overexpression of PrPc in cancer cell lines and the mechanisms involved in it.

The levels of PrPc in gastric cancer cell lines SGC7901, AGS, HepG2 and MKN28 were measured by semi-quantitative reverse transcription polymerase chain action (RT-PCR) under hypoxic and control conditions (Liang et al, 2007). PrPc activity in the MKN29 line showed the most hypoxic sensitivity, with western blot and immunoflourescent analysis showing PrPc expression to increase from 2h-8hrs after hypoxic exposure before returning to its basal level after 24hrs. These results showed that hypoxic induced PrPc activity occurs at mRNA and protein levels as well as a possible involvement in oxygen-regulated gene expression. The gastric cancer cell line, MKN28 was transfected with heat shock elements (HSE), luciferase constructs of the PrPc promoter. This was done to see where PrPc expression took place and its results showed higher luciferase activity in HSE containing cells, rather that those that has no HSE. This PrPc upregulation, however was depressed by MERK/ERK inhibitor (PD98059), a mitogen activated protein kinase (MAPK) that is activated in response to stresses such as hypoxia. Through these findings, Liang et al (2007) proposed that HSE in the promoter regions of PrPc and the transcription factors produced by ERK1/2, govern the regulation of PrPc expression in gastric cancer cell lines in hypoxia.


Whether the scrapie isoform, PrPsc and its toxic aggregates are the cause of cell death, or whether PrPc modifications, adding to the loss of function theory, cause prion disease pathogenesis has evoked much debate (Linden et al, 2008). PrPc has been showed to mediate neuroprotective responses, neuronal survival and development controlled by protein synthesis (Roffe et al., 2010). This link between PrPc and protein synthesis, which had not been previously explored, was addressed by Roffe et al (2010) who revealed that PrPc and stress inducible protein 1 (STI1) together increase protein synthesis in neurons. This PrPc-STI1 interaction, being depression the extent of protein synthesis, mediates neuroprotection and neuritogenesis via the PI3K-mTOR signalling pathway (Roffe et al., 2010). To validate the assumption whether that PrPc-STI1 interaction is directly involved in the regulation of proteins synthesis, hippocampal PrP neurons were metabolically labelled with [35S] - methionine, and its radioactivity was measured. Wildtype (WT) neurons (Prnp+/+) treated with STI1, showed a dose-dependent increase in protein synthesis, whilst PrPc null neurons (Prnp0/0) didn't produce a response. This shows that STI1 signalling is dependant of PrPc. Analysis of translation initiation levels showed a 1.4 ratio of untreated polysome whereas neurons treated with STI1 had a ratio of 2.9, suggesting that PrPc-STI1 increases protein synthesis via stimulating translation. The pathways taken to achieve protein synthesis by PrPc-STI1 interaction were studied by pre-treating hippocampal neurons with inhibitors of the PI3k, mTORC1 and ERK1/2 pathways (LY294002, rapamycin and PD98059 respectively). This inhibition repressed STI1 induced protein synthesis. In supporting the PI3k-Akt-mTOR and ERK1/2 pathways involvement in protein synthesis by PrPc-STI1 binding, phosphorylation values of their downstream targets were evaluated. Akt was rapidly phosphorylated in WT cells, as was P7056K, a target of mTOR. In the PrPc null cells, no increase in phosphorylation was observed. The 4E-BP's (4E-BP1, 4E-BP2, 4E-BP3) are also targets of the mTORC1 pathway with 4E-BP2 being the main neuronally expressed target. 4E-BP2, when treated with STI1, produced an increase in phosphorylation in WT, but not PrPc null neurons, as did ERK1/2's translation initiation factor eIF4E. mTOR's downstream targets, Akt, P7056K and 4E-BP2 mediate protein synthesis resulting in increased phosphorylation, implicating PrPc-STI1's involvement in this process. The importance of these findings for neuronal processes is its role in neuritogenesis and neuroprotection. In WT (Prnp+/+) hippocampal neurons PrPc-STI1 interactions, initiated neurite cell growth /9and therefore neuritogenesis), the effects of which were abolished when pre-treated with mTOR pathway inhibitors LY29002 and rapamycin, PrPc-STI1 interactions induced neuroprotective activity which was impaired when the previously mentioned inhibitors were applied.

Altogether, this data suggests that neuritogenesis and neuroprotection, resulting from PrPcSTI1 interactions are dependent upon protein synthesis via the mTOR pathway.


The prion protein, PrP, plays a major role in the pathogenesis of prion disease although the physiological function, by which its normal isoform, PrPc, works, is still somewhat unknown. Studies have shown that PrPc overexpression produces a phenotype more resistant to oxidative stress than PrPc null cells (Choi et al., 2007). This overexpression is present whether induced by manganese or copper, providing a means of protection as the prion protein interfering with the metals uptake (Choi et al., 2007). Oxidative stress and the resulting accumulation of reactive oxygen species, due to high Cu(II) levels, indicate PrPc's role in combating copper toxicity. Other studies showing the upregulation of PrPc in peri-infarcted brain tissue and patients suffering from ischemic stroke, suggest that the modifications of PrPc levels could prove beneficial for possible treatment of stroke (Mitsios et al., 2007)

Ultimately, understanding the mechanisms and physiological function of PrPc is essential in not only understanding the pathogenesis of prion and other related diseases, but also potential therapeutic strategies in treating these neurological conditions.