Quantitative imaging of tissue sections using infrared scanning technology.

Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi-quantitative results. Visualising infrared (IR) 'tags', with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR-based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR-based scanning technology yields comparable area-based quantification to those obtained from a modern high-resolution digital slide scanner. Secondly, IR-based dual target visualisation and expression-based quantification is rapid and simple. Thirdly, IR-based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR-based QFIHC provides an alternative method of rapid whole-tissue section low-resolution imaging for the production of reliable and accurate quantitative data.

PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the "prion-like" spread of aggregated forms of the beta-amyloid peptide (Aß), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre-formed fibrils may, therefore, more closely resemble a seeded proteinopathy than an infectious TSE disease.

Insights into Mechanisms of Chronic Neurodegeneration.

Chronic neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and prion diseases are characterised by the accumulation of abnormal conformers of a host encoded protein in the central nervous system. The process leading to neurodegeneration is still poorly defined and thus development of early intervention strategies is challenging. Unique amongst these diseases are Transmissible Spongiform Encephalopathies (TSEs) or prion diseases, which have the ability to transmit between individuals. The infectious nature of these diseases has permitted in vivo and in vitro modelling of the time course of the disease process in a highly reproducible manner, thus early events can be defined. Recent evidence has demonstrated that the cell-to-cell spread of protein aggregates by a "prion-like mechanism" is common among the protein misfolding diseases. Thus, the TSE models may provide insights into disease mechanisms and testable hypotheses for disease intervention, applicable to a number of these chronic neurodegenerative diseases.

Membrane pathology and microglial activation of mice expressing membrane anchored or membrane released forms of Aß and mutated human Alzheimer's precursor protein (APP).

AIMS: Alzheimer's disease and the transmissible spongiform encephalopathies or prion diseases accumulate misfolded and aggregated forms of neuronal cell membrane proteins. Distinctive membrane lesions caused by the accumulation of disease-associated prion protein (PrP(d)) are found in prion disease but morphological changes of membranes are not associated with Aß in Alzheimer's disease. Membrane changes occur in all prion diseases where PrP(d) is attached to cell membranes by a glycosyl-phosphoinositol (GPI) anchor but are absent from transgenic mice expressing anchorless PrP(d). Here we investigate whether GPI membrane attached Aß may also cause prion-like membrane lesions. METHODS: We used immunogold electron microscopy to determine the localization and pathology of Aß accumulation in groups of transgenic mice expressing anchored or unanchored forms of Aß or mutated human Alzheimer's precursor protein. RESULTS: GPI attached Aß did not replicate the membrane lesions of PrP(d). However, as with PrP(d) in prion disease, Aß peptides derived from each transgenic mouse line initially accumulated on morphologically normal neurite membranes, elicited rapid glial recognition and neurite Aß was transferred to attenuated microglial and astrocytic processes. CONCLUSIONS: GPI attachment of misfolded membrane proteins is insufficient to cause prion-like membrane lesions. Prion disease and murine Aß amyloidosis both accumulate misfolded monomeric or oligomeric membrane proteins that are recognized by glial processes and acquire such misfolded proteins prior to their accumulation in the extracellular space. In contrast to prion disease where glial cells efficiently endocytose PrP(d) to endolysosomes, activated microglial cells in murine Aß amyloidosis are not as efficient phagocytes.

Molecular barriers to zoonotic transmission of prions.

The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.

Increased susceptibility of transgenic mice expressing human PrP to experimental sheep bovine spongiform encephalopathy is not due to increased agent titre in sheep brain tissue.

Bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt-Jakob disease in humans have previously been shown to be caused by the same strain of transmissible spongiform encephalopathy agent. It is hypothesized that the agent spread to humans following consumption of food products prepared from infected cattle. Despite evidence supporting zoonotic transmission, mouse models expressing human prion protein (HuTg) have consistently shown poor transmission rates when inoculated with cattle BSE. Higher rates of transmission have however been observed when these mice are exposed to BSE that has been experimentally transmitted through sheep or goats, indicating that humans may potentially be more susceptible to BSE from small ruminants. Here we demonstrate that increased transmissibility of small ruminant BSE to HuTg mice was not due to replication of higher levels of infectivity in sheep brain tissue, and is instead due to other specific changes in the infectious agent.

Dissociation between transmissible spongiform encephalopathy (TSE) infectivity and proteinase K-resistant PrP(Sc) levels in peripheral tissue from a murine transgenic model of TSE disease.

Most current diagnostic tests for transmissible spongiform encephalopathies (TSE) rely on the presence of proteinase K (PK)-resistant PrP(Sc) (PrP-res) in postmortem tissues as an indication of TSE disease. However, a number of studies have highlighted a discrepancy between TSE infectivity and PrP-res levels in both natural and experimental cases of TSE disease. Previously, we have shown high TSE infectivity levels in the brain tissue of mice that have a clinical TSE disease with associated vacuolar pathology but little or no detectable PrP-res. Here, the levels of TSE infectivity and PrP-res within a peripheral tissue of this mouse model were investigated. Biochemical analysis showed that low levels of PrP-res were present in the spleen tissue in comparison to the levels observed in the spleen of mice infected with ME7 or 79A. However, upon subpassage of brain and spleen tissue from clinically ill mice with little or no PrP-res detectable, similar short incubation periods to disease were observed, indicating that infectivity levels were similarly high in both tissues. Thus, the discrepancy between PrP-res and TSE infectivity was also present in the peripheral tissues of this disease model. This result indicates that peripheral tissues can contain higher levels of infectivity given the correct combination of host species, PrP genotype, and TSE agent. Therefore, the assumption that the levels of peripheral infectivity are lower than those in the central nervous system is not always correct, and this could have implications for current food safety regulations.

Dissociation of prion protein amyloid seeding from transmission of a spongiform encephalopathy.

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar "prion-like" mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer's disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP(TSE)) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP(TSE) from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP(TSE). In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP(TSE) is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP(TSE) in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.

Characterization of an unusual transmissible spongiform encephalopathy in goat by transmission in knock-in transgenic mice.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle, and its transmission to humans through contaminated food is thought to be the cause of the variant form of Creutzfeldt-Jakob disease. BSE is believed to have spread from the recycling in cattle of ruminant tissue in meat and bone meal (MBM). However, during this time, sheep and goats were also exposed to BSE-contaminated MBM. Both sheep and goats are experimentally susceptible to BSE, and while there have been no reported natural BSE cases in sheep, two goat BSE field cases have been documented. While cases of BSE are rare in small ruminants, the existence of scrapie in both sheep and goats is well established. In the UK, during 2006-2007, a serious outbreak of clinical scrapie was detected in a large dairy goat herd. Subsequently, 200 goats were selected for post-mortem examination, one of which showed biochemical and immunohistochemical features of the disease-associated prion protein (PrP(TSE)) which differed from all other infected goats. In the present study, we investigated this unusual case by performing transmission bioassays into a panel of mouse lines. Following characterization, we found that strain properties such as the ability to transmit to different mouse lines, lesion profile pattern, degree of PrP deposition in the brain and biochemical features of this unusual goat case were neither consistent with goat BSE nor with a goat scrapie herdmate control. However, our results suggest that this unusual case has BSE-like properties and highlights the need for continued surveillance.

Presence of subclinical infection in gene-targeted human prion protein transgenic mice exposed to atypical bovine spongiform encephalopathy.

The transmission of bovine spongiform encephalopathy (BSE) to humans, leading to variant Creutzfeldt-Jakob disease has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health. Until recently, TSE disease in cattle was thought to be caused by a single agent strain, BSE, also known as classical BSE, or BSE-C. However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. To model the risk to human health, we previously inoculated these two forms of atypical BSE (BASE and BSE-H) into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP) (HuTg) but were unable to detect any signs of TSE pathology in these mice. However, despite the absence of TSE pathology, upon subpassage of some BASE-challenged HuTg mice, a TSE was observed in recipient gene-targeted bovine PrP Tg (Bov6) mice but not in HuTg mice. Disease transmission from apparently healthy individuals indicates the presence of subclinical BASE infection in mice expressing human PrP that cannot be identified by current diagnostic methods. However, due to the lack of transmission to HuTg mice on subpassage, the efficiency of mouse-to-mouse transmission of BASE appears to be low when mice express human rather than bovine PrP.

Bovine PrP expression levels in transgenic mice influence transmission characteristics of atypical bovine spongiform encephalopathy.

Until recently, transmissible spongiform encephalopathy (TSE) disease in cattle was thought to be caused by a single agent strain, bovine spongiform encephalopathy (BSE) (classical BSE or BSE-C). However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. These atypical BSE isolates have been previously transmitted to a range of transgenic mouse models overexpressing PrP from different species at different levels, on a variety of genetic backgrounds. To control for genetic background and expression level in the analysis of these isolates, we performed here a comprehensive comparison of the neuropathological and molecular properties of all three BSE agents (BASE, BSE-C and BSE-H) upon transmission into the same gene-targeted transgenic mouse line expressing the bovine prion protein (Bov6) and a wild-type control of the same genetic background. Significantly, upon challenge with these BSE agents, we found that BASE did not produce shorter survival times in these mice compared with BSE-C, contrary to previous studies using overexpressing bovine transgenic mice. Amyloid plaques were only present in mice challenged with atypical BSE and neuropathological features, including intensity of PrP deposition in the brain and severity of vacuolar degeneration were less pronounced in BASE compared with BSE-C-challenged mice.

Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein.

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep.

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.

Glycosylation of PrPC determines timing of neuroinvasion and targeting in the brain following transmissible spongiform encephalopathy infection by a peripheral route.

Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the periphery to the central nervous system where pathological lesions are formed. Several routes and cells within the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein cellular PrP (PrP(C)) is considered a key factor for replication of infectivity in the central nervous system (CNS) and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms of PrP have in these processes remain to be determined. To address this issue, we have used our unique models of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoculated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the glycosylation status of host PrP.

Host PrP glycosylation: a major factor determining the outcome of prion infection.

The expression of the prion protein (PrP) is essential for transmissible spongiform encephalopathy (TSE) or prion diseases to occur, but the underlying mechanism of infection remains unresolved. To address the hypothesis that glycosylation of host PrP is a major factor influencing TSE infection, we have inoculated gene-targeted transgenic mice that have restricted N-linked glycosylation of PrP with three TSE strains. We have uniquely demonstrated that mice expressing only unglycosylated PrP can sustain a TSE infection, despite altered cellular location of the host PrP. Moreover we have shown that brain material from mice infected with TSE that have only unglycosylated PrP(Sc) is capable of transmitting infection to wild-type mice, demonstrating that glycosylation of PrP is not essential for establishing infection within a host or for transmitting TSE infectivity to a new host. We have further dissected the requirement of each glycosylation site and have shown that different TSE strains have dramatically different requirements for each of the glycosylation sites of host PrP, and moreover, we have shown that the host PrP has a major role in determining the glycosylation state of de novo generated PrP(Sc).

Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures.

Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures is an on-going question. Here, we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines.

Molecular model of prion transmission to humans.

To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro. Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.

Differential protein profiling as a potential multi-marker approach for TSE diagnosis.

BACKGROUND: Transmissible spongiform encephalopathy describes a family of diseases affecting both man and animals. Current tests for the diagnosis of these diseases are based on the detection of an abnormal misfolded form of the host protein PrP which is found within the central nervous and lymphoreticular systems of affected animals. Recently, concern that this marker may not be as reliable as previously thought, coupled with an urgentneed for a pre-clinical live animal test, has led to the search for alternative assays for the detection of TSE disease. METHODS: This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF) for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers.Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. RESULTS: Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. CONCLUSION: Differential protein expression profiling has the potential to be used for the detection of disease in TSE infected animals. Having established that a "training set" of potential markers can be constructed, more work would be required to further test the specificity and sensitivity of the assay in a "testing set". Based on these promising results, further studies are being performed using blood samples from infected sheep to assess the potential use of SELDI-TOF as a pre-mortem blood based diagnostic.

The role of host PrP in Transmissible Spongiform Encephalopathies.

PrP has a central role in the Transmissible Spongiform Encephalopathies (TSEs), and mutations and polymorphisms in host PrP can profoundly alter the host's susceptibility to a TSE agent. However, precisely how host PrP influences the outcome of disease has not been established. To investigate this we have produced by gene targeting a series of inbred lines of transgenic mice expressing different PrP genes. This allows us to study directly the influence of the host PrP gene in TSEs. We have examined the role of glycosylation, point mutations, polymorphisms and PrP from different species on host susceptibility and the disease process both within the murine species and across species barriers.

Infectious prions and proteinopathies.

Transmissible spongiform encephalopathies (TSEs) are caused by an infectious agent that is thought to consist of only misfolded and aggregated prion protein (PrP). Unlike conventional micro-organisms, the agent spreads and propagates by binding to and converting normal host PrP into the abnormal conformer, increasing the infectious titre. Synthetic prions, composed of refolded fibrillar forms of recombinant PrP (rec-PrP) have been generated to address whether PrP aggregates alone are indeed infectious prions. In several reports, the development of TSE disease has been described following inoculation and passage of rec-PrP fibrils in transgenic mice and hamsters. However in studies described here we show that inoculation of rec-PrP fibrils does not always cause clinical TSE disease or increased infectious titre, but can seed the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). These data are reminiscent of the "prion-like" spread of misfolded protein in other models of neurodegenerative disease following inoculation of transgenic mice with pre-formed amyloid seeds. Protein misfolding, even when the protein is PrP, does not inevitably lead to the development of an infectious TSE disease. It is possible that most in vivo and in vitro produced misfolded PrP is not infectious and that only a specific subpopulation is associated with infectivity and neurotoxicity.