Introduction to Current Progress in Advanced Research on Prions.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurological diseases that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, camel spongiform encephalopathy (CSE) in camels and chronic wasting disease (CWD) in cervids. A key event in prion diseases is the conversion of the cellular, host-encoded prion protein (PrPC) to its abnormal isoform (PrPSc) predominantly in the central nervous system of the infected host (Aguzzi et al., 2004). These diseases are transmissible under some circumstances, but unlike other transmissible disorders, prion diseases can also be caused by mutations in the host gene. The mechanism of prion spread among sheep and goats that develop natural scrapie is unknown. CWD, transmissible mink encephalopathy (TME), BSE, feline spongiform encephalopathy (FSE), and exotic ungulate encephalopathy (EUE) are all thought to occur after the consumption of prion-infected material. Most cases of human prion disease occur from unknown reasons, and greater than 20 mutations in the prion protein (PrP) gene may lead to inherited prion disease. In other instances, prion diseases are contracted by exposure to prion infectivity. These considerations raise the question of how a mere protein aggregate can bypass mucosal barriers, circumvent innate and adoptive immunity, and traverse the blood-brain barrier to give rise to brain disease. Here, we will briefly introduce a few topics in current prion studies.

Mitochondrial dysfunction in preclinical genetic prion disease: A target for preventive treatment?

Mitochondrial malfunction is a common feature in advanced stages of neurodegenerative conditions, as is the case for the accumulation of aberrantly folded proteins, such as PrP in prion diseases. In this work, we investigated mitochondrial activity and expression of related factors vis a vis PrP accumulation at the subclinical stages of TgMHu2ME199K mice, modeling for genetic prion diseases. While these mice remain healthy until 5-6 months of age, they succumb to fatal disease at 12-14 months. We found that mitochondrial respiratory chain enzymatic activates and ATP/ROS production, were abnormally elevated in asymptomatic mice, concomitant with initial accumulation of disease related PrP. In parallel, the expression of Cytochrome c oxidase (COX) subunit IV isoform 1(Cox IV-1) was reduced and replaced by the activity of Cox IV isoform 2, which operates in oxidative neuronal conditions. At all stages of disease, Cox IV-1 was absent from cells accumulating disease related PrP, suggesting that PrP aggregates may directly compromise normal mitochondrial function. Administration of Nano-PSO, a brain targeted antioxidant, to TgMHu2ME199K mice, reversed functional and biochemical mitochondrial functions to normal conditions regardless of the presence of misfolded PrP. Our results therefore indicate that in genetic prion disease, oxidative damage initiates long before clinical manifestations. These manifest only when aggregated PrP levels are too high for the compensatory mechanisms to sustain mitochondrial activity.

Ganglioside Synthase Knockout Reduces Prion Disease Incubation Time in Mouse Models.

Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.

Prion infectivity is encoded exclusively within the structure of proteinase K-resistant fragments of synthetically generated recombinant PrPSc.

Transmissible spongiform encephalopathies, also known as prion diseases, are a group of fatal neurodegenerative disorders affecting both humans and animals. The central pathogenic event in prion disease is the misfolding of normal prion protein (PrPC) into the pathogenic conformer, PrPSc, which self-replicates by converting PrPC to more of itself. The biochemical hallmark of PrPSc is its C-terminal resistance to proteinase K (PK) digestion, which has been historically used to define PrPSc and is still the most widely used characteristic for prion detection. We used PK-resistance as a biochemical measure for the generation of recombinant prion from bacterially expressed recombinant PrP. However, the existence of both PK- resistant and -sensitive PrPSc forms in animal and human prion disease led to the question of whether the in vitro-generated recombinant prion infectivity is due to the PK-resistant or -sensitive recombinant PrP forms. In this study, we compared undigested and PK-digested recombinant prions for their infectivity using both the classical rodent bioassay and the cell-based prion infectivity assay. Similar levels of infectivity were detected in PK-digested and -undigested samples by both assays. A time course study of recombinant prion propagation showed that the increased capability to seed the conversion of endogenous PrP in cultured cells coincided with an increase of the PK-resistant form of recombinant PrP. Moreover, prion infectivity diminished when recombinant prion was subjected to an extremely harsh PK digestion. These results demonstrated that the infectivity of recombinant prion is encoded within the structure of the PK-resistant PrP fragments. This characteristic of recombinant prion, that a simple PK digestion is able to eliminate all PK-sensitive (non-infectious) PrP species, makes possible a more homogenous material that will be ideal for dissecting the molecular basis of prion infectivity.

Role of the AMPK pathway in promoting autophagic flux via modulating mitochondrial dynamics in neurodegenerative diseases: Insight into prion diseases.

Neurons are highly energy demanding cells dependent on the mitochondrial oxidative phosphorylation system. Mitochondria generate energy via respiratory complexes that constitute the electron transport chain. Adenosine triphosphate depletion or glucose starvation act as a trigger for the activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK is an evolutionarily conserved protein that plays an important role in cell survival and organismal longevity through modulation of energy homeostasis and autophagy. Several studies suggest that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. Mild mitochondrial dysfunction leads to activated AMPK signaling, but severe endoplasmic reticulum stress and mitochondrial dysfunction may lead to a shift from autophagy towards apoptosis and perturbed AMPK signaling. Hence, controlling mitochondrial dynamics and autophagic flux via AMPK activation might be a useful therapeutic strategy in neurodegenerative diseases to reinstate energy homeostasis and degrade misfolded proteins. In this review article, we discuss briefly the role of AMPK signaling in energy homeostasis, the structure of AMPK, activation mechanisms of AMPK, regulation of AMPK, the role of AMPK in autophagy, the role of AMPK in neurodegenerative diseases, and finally the role of autophagic flux in prion diseases.

Uncontrolled SFK-mediated protein trafficking in prion and Alzheimer's disease.

Prions and Amyloid beta (Aß) peptides induce synaptic damage via complex mechanisms that include the pathological alteration of intracellular signaling cascades. The host-encoded cellular prion protein (PrPC) acts as a high-affinity cell surface receptor for both toxic species and it can modulate the endocytic trafficking of the N-methyl D-aspartate (NMDA) receptor and E-cadherin adhesive complexes via Src family kinases (SFKs). Interestingly, SFK-mediated control of endocytosis is a widespread mechanism used to regulate the activity of important transmembrane proteins, including neuroreceptors for major excitatory and inhibitory neurotransmitters. Here we discuss our recent work in zebrafish and accumulating evidence suggesting that subversion of this pleiotropic regulatory mechanism by Aß oligomers and prions explains diverse neurotransmission deficits observed in human patients and mouse models of prion and Alzheimer's neurodegeneration. While Aß, PrPC and SFKs constitute potential therapeutic targets on their own, drug discovery efforts might benefit significantly from aiming at protein-protein interactions that modulate the endocytosis of specific SFK targets.

Phosphatidylinositol-glycan-phospholipase D is involved in neurodegeneration in prion disease.

PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer's disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.

Regulation of proteasomes in prion disease.

The hallmark of prion disease is the accumulation of misfolded protein PrP(Sc), which is toxic to neuronal cells. The proteasome system is responsible for the rapid, precise, and timely degradation of proteins and plays an important role in cellular protein quality control. Increasing evidence indicates impaired activity of proteasomes in prion diseases. Accumulated PrP(Sc) can directly or indirectly affect proteasome activity. Misfolded protein may influence the assembly and activity of 19S regulatory particle, or post-translational modification of 20S proteasome, which may adversely affect the protein degradation activity of proteasomes. In this review, we summarized the recent findings concerning the possible regulation of proteasomes in prion and other neurodegenerative diseases. The proteasome system may enhance its degradation activity by changing its structure, and this activity can also be increased by related chaperones when neuronal cells are subject to stress. When the proteasome system is inhibited, degradation of protein aggregates via autophagy may increase as a compensatory system. It is possible that a balance exists between the proteasome and autophagy in vivo; when one is impaired, the activity of the other may increase to maintain homeostasis. However, more studies are needed to elucidate the relationship between the proteasome system and autophagy.

Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease.

Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced proteolytic activation of PKCδ in our COSC model. Immunohistochemical analysis of scrapie-infected COSC revealed loss of PKCδ positive Purkinje cells and enhanced astrocyte proliferation. Further examination of PKCδ signaling in the RML scrapie adopted in vivo mouse model showed increased proteolytic cleavage and Tyr 311 phosphorylation of the kinase. Notably, we observed a delayed onset of scrapie-induced motor symptoms in PKCδ knockout (PKCδ(-/-)) mice as compared with wild-type (PKCδ(+/+)) mice, further substantiating the role of PKCδ in prion disease. Collectively, these data suggest that PKCδ signaling likely plays a role in the neurodegenerative processes associated with prion diseases.

Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease.

AIMS: Transmissible spongiform encephalopathies, also called prion diseases, are characterized by the cerebral accumulation of misfolded prion protein (PrP(SC) ) and subsequent neurodegeneration. However, despite considerable research effort, the molecular mechanisms underlying prion-induced neurodegeneration are poorly understood. Here, we explore the hypothesis that prions induce dysfunction of the PI3K/Akt/GSK-3 signalling pathway. METHODS: We employed two parallel approaches. Using cell cultures derived from mouse primary neurones and from a human neuronal cell line, we identified common elements that were modified by the neurotoxic fragment of PrP(106-126) . These studies were then complemented by comparative analyses in a mouse model of prion infection. RESULTS: The presence of a polymerized fragment of the prion protein (PrP(106-126) ) or of a prion strain altered PI3K-mediated signalling, as evidenced by Akt inhibition and GSK-3 activation. PI3K activation by the addition of insulin or the expression of a constitutively active Akt mutant restored normal levels of Akt and GSK-3 activity. These changes were correlated with a reduction in caspase activity and an increase in neuronal survival. Moreover, we found that activation of caspase 3, Erk and GSK-3 are common features of PrP(106-126) -mediated neurotoxicity in cellular systems and prion infection in the mouse cerebellum, while activation of caspase 12 and JNK was observed in cellular models. CONCLUSIONS: Our findings in cell culture and in vivo models of prion disease demonstrate marked alterations to the PI3K/Akt/GSK-3 pathway and suggest that two additional pathways contribute to PrP-induced neurotoxicity as responsible of JNK and caspase 12 activation.

Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.

During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA-like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated.

Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008.

Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified prospectively in the U.K. since the first description of the condition in 2008 in the U.S.A. To determine the incidence and phenotype of variably protease-sensitive prionopathy within a single well-defined cohort, we have conducted a retrospective review of patients referred to the National Creutzfeldt-Jakob Disease Research & Surveillance Unit during the period 1991-2008. The approach taken was to screen frozen brain tissue by western blotting for the form of protease-resistant prion protein that characterizes variably protease-sensitive prionopathy, followed by neuropathological and clinical review of candidate cases. Cases diagnosed as sporadic Creutzfeldt-Jakob disease with atypical neuropathology were also reviewed. Four hundred and sixty-five cases were screened biochemically, yielding four candidate cases of variably protease-sensitive prionopathy. One was discounted on pathological and clinical grounds, and one was a known case of variably protease-sensitive prionopathy previously reported, leaving two new cases, which were confirmed biochemically and neuropathologically as variably protease-sensitive prionopathy. A third new case that lacked frozen tissue was recognized retrospectively on neuropathological grounds alone. This means that five cases of variably protease-sensitive prionopathy have been identified (prospectively and retrospectively) during the surveillance period 1991-2011 in the U.K. Assuming ascertainment levels equivalent to that of other human prion diseases, these data indicate that variably protease-sensitive prionopathy is a rare phenotype within human prion diseases, which are themselves rare. Biochemical investigation indicates that the abnormal protease-resistant prion protein fragment that characterizes variably protease-sensitive prionopathy is detectable at low levels in some cases of sporadic Creutzfeldt-Jakob disease and conversely, that the form of abnormal prion protein that characterizes sporadic Creutzfeldt-Jakob disease can be found in certain brain regions of cases of variably protease-sensitive prionopathy, indicating molecular overlaps between these two disorders.

Roles of endoproteolytic α-cleavage and shedding of the prion protein in neurodegeneration.

The cellular prion protein (PrP(C)) plays important roles in neurodegenerative diseases. First, it is the well-established substrate for the conformational conversion into its pathogenic isoform (PrP(Sc)) giving rise to progressive and fatal prion diseases. Moreover, several recent reports highlight important roles of PrP(C) in other neurodegenerative conditions such as Alzheimer's disease. Since PrP(C) is subject to proteolytic processing, here we discuss the two main cleavage events under physiological conditions, α-cleavage and shedding. We focus on how these cleavages and the resulting fragments may impact prion diseases as well as other neurodegenerative proteinopathies. Finally, we discuss the recently identified sheddase of PrP(C), namely the metalloprotease ADAM10, with regard to therapeutic potential against neurodegenerative diseases.

Peptidylarginine deiminase and protein citrullination in prion diseases: strong evidence of neurodegeneration.

The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.

A new mechanism for transmissible prion diseases.

The transmissible agent of prion disease consists of prion protein (PrP) in ß-sheet-rich state (PrP(Sc)) that can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrP(Sc) template. Here, three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrP(Sc), whereas an alternative amyloid state, with a folding pattern different from that of PrP(Sc), induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrP(Sc) emerged. The dynamics between the two forms suggest that atypical proteinase K-resistant PrP (PrPres) gave rise to PrP(Sc). While no PrP(Sc) was found in preparations of fibrils using protein misfolding cyclic amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb, suggesting that atypical PrPres was the first product of PrP(C) misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrP(Sc)-templated or spontaneous conversion of PrP(C) into PrP(Sc) exists. This study provides compelling evidence that noninfectious amyloids with a structure different from that of PrP(Sc) could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

Role of cyclophilin A from brains of prion-infected mice in stimulation of cytokine release by microglia and astroglia in vitro.

Prion diseases or transmissible spongiform encephalopathy diseases are typically characterized by deposition of abnormally folded partially protease-resistant host-derived prion protein (PrPres), which is associated with activated glia and increased release of cytokines. This neuroinflammatory response may play a role in transmissible spongiform encephalopathy pathogenesis. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing IL-12p40 and CCL2 (MCP-1), glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or prion protein amyloid fibrils produced in vitro. However, significant glial stimulation was induced by clarified scrapie brain homogenates lacking PrPres. This stimulation was greatly reduced both by antibody to cyclophilin A (CyPA), a known mediator of inflammation in peripheral tissues, and by cyclosporine A, a CyPA inhibitor. In biochemical studies, purified truncated CyPA fragments stimulated a pattern of cytokine release by microglia and astroglia similar to that induced by scrapie-infected brain homogenates, whereas purified full-length CyPA was a poor stimulator. This requirement for CyPA truncation was not reported in previous studies of stimulation of peripheral macrophages, endothelial cell cardiomyocytes, and vascular smooth muscle cells. Therefore, truncated CyPA detected in brain following prion infection may have an important role in the activation of brain-derived primary astroglia and microglia in prion disease and perhaps other neurodegenerative or neuroinflammatory diseases.

Protease-resistant PrP and PrP oligomers in the brain in human prion diseases after intraventricular pentosan polysulfate infusion.

Intraventricular infusion of pentosan polysulfate (PPS) as a treatment for various human prion diseases has been applied in Japan. To evaluate the influence of PPS treatment we performed pathological examination and biochemical analyses of PrP molecules in autopsied brains treated with PPS (one case of sporadic Creutzfeldt-Jakob disease (sCJD, case 1), two cases of dura mater graft-associated CJD (dCJD, cases 2 and 4), and one case of Gerstmann-Sträussler-Scheinker disease (GSS, case 3). Six cases of sCJD without PPS treatment were examined for comparison. Protease-resistant PrP (PrP(res) ) in the frontal lobe was evaluated by Western blotting after proteinase K digestion. Further, the degree of polymerization of PrP molecules was examined by the size-exclusion gel chromatography assay. PPS infusions were started 3-10 months after disease onset, but the treatment did not achieve any clinical improvements. Postmortem examinations of the treated cases revealed symmetrical brain lesions, including neuronal loss, spongiform change and gliosis. Noteworthy was GFAP in the cortical astrocytes reduced in all treated cases despite astrogliosis. Immunohistochemistry for PrP revealed abnormal synaptic deposits in all treated cases and further plaque-type PrP deposition in case 3 of GSS and case 4 of dCJD. Western blotting showed relatively low ratios of PrP(res) in case 2 of dCJD and case 3 of GSS, while in the treated sCJD (case 1), the ratio of PrP(res) was comparable with untreated cases. The indices of oligomeric PrP were reduced in one sCJD (case 1) and one dCJD (case 2). Although intraventricular PPS infusion might modify the accumulation of PrP oligomers in the brains of patients with prion diseases, the therapeutic effects are still uncertain.

Optical imaging detects apoptosis in the brain and peripheral organs of prion-infected mice.

Activation of the caspase family of cysteine proteases is proposed to be an important cell death mechanism in transmissible spongiform encephalopathies or prion diseases. We determined the extent of caspase activation in the brain and peripheral organs of mice that showed clinical signs after intracerebral inoculation with mouse-adapted prions by in vivo administration of a red fluorescent pan-caspase inhibitor, sulforhodamine B-Val-Ala-Asp(OMe)-fluoromethylketone. Fluorescence reflectance imaging identified a significant increase in active caspases in brains of prion-infected, but not uninfected, mice that correlated with increases in procaspase-3 and cleaved caspase-3, a central effector caspase, assessed by Western immunoblot analysis. Fluorescence was found in brain regions in which neuronal loss occurs; immunohistochemical analysis indicated that fluorescence was localized within and adjacent to deposits of abnormal disease-associated conformers of the prion protein (PrP Sc). Fluorescence was also significantly increased in the kidney, lung, and ileum of prion-infected mice. This premortem labeling of caspase activation in the brain, and importantly in peripheral organs, could be exploited as a biomarker for longitudinal monitoring of prion disease progression and the impact of therapy in vivo in addition to, or independently of, PrP and spongiform changes.

Variably protease-sensitive prionopathy: a new sporadic disease of the prion protein.

OBJECTIVE: The objective of the study is to report 2 new genotypic forms of protease-sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV). METHODS: Fifteen affected subjects with 129MM, 129MV, and 129VV underwent comparative evaluation at the National Prion Disease Pathology Surveillance Center for clinical, histopathologic, immunohistochemical, genotypical, and PrP characteristics. RESULTS: Disease duration (between 22 and 45 months) was significantly different in the 129VV and 129MV subjects. Most other phenotypic features along with the PrP electrophoretic profile were similar but distinguishable in the 3 129 genotypes. A major difference laid in the sensitivity to protease digestion of the disease-associated PrP, which was high in 129VV but much lower, or altogether lacking, in 129MV and 129MM. This difference prompted the substitution of the original designation with "variably protease-sensitive prionopathy" (VPSPr). None of the subjects had mutations in the PrP gene coding region. INTERPRETATION: Because all 3 129 genotypes are involved, and are associated with distinguishable phenotypes, VPSPr becomes the second sporadic prion protein disease with this feature after Creutzfeldt-Jakob disease, originally reported in 1920. However, the characteristics of the abnormal prion protein suggest that VPSPr is different from typical prion diseases, and perhaps more akin to subtypes of Gerstmann-Sträussler-Scheinker disease.