Microglia are not required for prion-induced retinal photoreceptor degeneration.

Degeneration of photoreceptors in the retina is a major cause of blindness in humans. Often retinal degeneration is due to inheritance of mutations in genes important in photoreceptor (PR) function, but can also be induced by other events including retinal trauma, microvascular disease, virus infection or prion infection. The onset of apoptosis and degeneration of PR neurons correlates with invasion of the PR cellular areas by microglia or monocytes, suggesting a causal role for these cells in pathogenesis of PR degenerative disease. To study the role of microglia in prion-induced retinal disease, we fed prion-infected mice a CSF-1 receptor blocking drug, PLX5622, to eliminate microglia in vivo, and the effects on retinal degeneration were analyzed over time. In mice not receiving drug, the main inflammatory cells invading the degenerating PR areas were microglia, not monocytes. Administration of PLX5622 was highly effective at ablating microglia in retina. However, lack of microglia during prion infection did not prevent degeneration of PR cells. Therefore, microglia were not required for the PR damage process during prion infection. Indeed, mice lacking microglia had slightly faster onset of PR damage. Similar results were seen in C57BL/10 mice and transgenic mice expressing GFP or RFP on microglia and monocytes, respectively. These results were supported by experiments using prion-infected Cx3cr1 knockout mice without PLX5622 treatment, where microglial expansion in retina was delayed, but PR degeneration was not. Contrary to predictions, microglia were not a causative factor in retinal damage by prion infection. Instead, newly generated PrPSc accumulated around the inner segment region of the PR cells and appeared to correlate with initiation of the pathogenic process in the absence of microglia.

Soluble Aß aggregates can inhibit prion propagation.

Mammalian prions cause lethal neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) and consist of multi-chain assemblies of misfolded cellular prion protein (PrPC). Ligands that bind to PrPC can inhibit prion propagation and neurotoxicity. Extensive prior work established that certain soluble assemblies of the Alzheimer's disease (AD)-associated amyloid ß-protein (Aß) can tightly bind to PrPC, and that this interaction may be relevant to their toxicity in AD. Here, we investigated whether such soluble Aß assemblies might, conversely, have an inhibitory effect on prion propagation. Using cellular models of prion infection and propagation and distinct Aß preparations, we found that the form of Aß assemblies which most avidly bound to PrP in vitro also inhibited prion infection and propagation. By contrast, forms of Aß which exhibit little or no binding to PrP were unable to attenuate prion propagation. These data suggest that soluble aggregates of Aß can compete with prions for binding to PrPC and emphasize the bidirectional nature of the interplay between Aß and PrPC in Alzheimer's and prion diseases. Such inhibitory effects of Aß on prion propagation may contribute to the apparent fall-off in the incidence of sporadic CJD at advanced age where cerebral Aß deposition is common.

Neurotoxicity of Prion Peptides on Cultured Cerebellar Neurons.

Prion neurotoxicity has been modeled in vitro using synthetic peptides derived from the PrPC sequence. The major region of neurotoxicity has been localized to the hydrophobic domain located in the middle of the PrP protein. Neurotoxicity assays are typically performed on cultured mouse cerebellar neurons derived from neonatal pups, and cell viability can be monitored by assays including MTT or MTS, cell death by LDH release, or apoptosis by caspase cleavage assays. These neurotoxicity studies have been useful in identifying cofactors, such as PrPC and metals, as modulators of PrP peptide-mediated neurotoxicity. Given the biosafety issues associated with handling and purifying infectious prions, the use of synthetic peptides, which display a dependence upon PrPC expression for toxicity, as per the PrPSc agent for infectivity, supports the relevance of using these synthetic peptides for understanding PrP-mediated neurotoxicity.

Pathogenic mechanisms of prion protein, amyloid-ß and α-synuclein misfolding: the prion concept and neurotoxicity of protein oligomers.

Proteinopathies represent a group of diseases characterized by the unregulated misfolding and aggregation of proteins. Accumulation of misfolded protein in the central nervous system (CNS) is associated with neurodegenerative diseases, such as the transmissible spongiform encephalopathies (or prion diseases), Alzheimer's disease, and the synucleinopathies (the most common of which is Parkinson's disease). Of these, the pathogenic mechanisms of prion diseases are particularly striking where the transmissible, causative agent of disease is the prion, or proteinaceous infectious particle. Prions are composed almost exclusively of PrPSc ; a misfolded isoform of the normal cellular protein, PrPC , which is found accumulated in the CNS in disease. Today, mounting evidence suggests other aggregating proteins, such as amyloid-ß (Aß) and α-synuclein (α-syn), proteins associated with Alzheimer's disease and synucleinopathies, respectively, share similar biophysical and biochemical properties with PrPSc that influences how they misfold, aggregate, and propagate in disease. In this regard, the definition of a 'prion' may ultimately expand to include other pathogenic proteins. Unifying knowledge of folded proteins may also reveal common mechanisms associated with other features of disease that are less understood, such as neurotoxicity. This review discusses the common features Aß and α-syn share with PrP and neurotoxic mechanisms associated with these misfolded proteins. Several proteins are known to misfold and accumulate in the central nervous system causing a range of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and the prion diseases. Prions are transmissible misfolded conformers of the prion protein, PrP, which seed further generation of infectious proteins. Similar effects have recently been observed in proteins associated with Alzheimer's disease and the synucleinopathies, leading to the proposition that the definition of a 'prion' may ultimately expand to include other pathogenic proteins. Unifying knowledge of misfolded proteins may also reveal common mechanisms associated with other features of disease that are less understood, such as neurotoxicity.

A Neuronal Culture System to Detect Prion Synaptotoxicity.

Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrPSc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrPC, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrPC molecule. Both protease-resistant and protease-sensitive forms of PrPSc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrPSc and testing potential therapeutic agents.

Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked.

Prions are lethal infectious agents thought to consist of multi-chain forms (PrP(Sc)) of misfolded cellular prion protein (PrP(C)). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrP(C) expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrP(C) expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrP(Sc) at a rate proportional to PrP(C) concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrP(C) concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrP(C) concentration dependent.

Using protein misfolding cyclic amplification generates a highly neurotoxic PrP dimer causing neurodegeneration.

Under the "protein-only" hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP(Sc). However, since PrP(Sc) is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP(Sc) may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP(Sc) by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23-231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.

Sustained translational repression by eIF2α-P mediates prion neurodegeneration.

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.

Prion propagation and toxicity in vivo occur in two distinct mechanistic phases.

Mammalian prions cause fatal neurodegenerative conditions including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. Prion infections are typically associated with remarkably prolonged but highly consistent incubation periods followed by a rapid clinical phase. The relationship between prion propagation, generation of neurotoxic species and clinical onset has remained obscure. Prion incubation periods in experimental animals are known to vary inversely with expression level of cellular prion protein. Here we demonstrate that prion propagation in brain proceeds via two distinct phases: a clinically silent exponential phase not rate-limited by prion protein concentration which rapidly reaches a maximal prion titre, followed by a distinct switch to a plateau phase. The latter determines time to clinical onset in a manner inversely proportional to prion protein concentration. These findings demonstrate an uncoupling of infectivity and toxicity. We suggest that prions themselves are not neurotoxic but catalyse the formation of such species from PrP(C). Production of neurotoxic species is triggered when prion propagation saturates, leading to a switch from autocatalytic production of infectivity (phase 1) to a toxic (phase 2) pathway.

A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein.

In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Delta105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Delta105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.

Role of microglia in age-related changes to the nervous system.

Microglia play a curious role in the nervous system. Their role is intrinsically protective and supportive, but during neurodegenerative disease, it is well established that microglia play a significant role in the initiation of neuronal death. Microglia, like neurons, show age-related changes that could potentially alter their behavior. While extreme changes to a large population of microglia cause dramatic neuronal loss in neurodegeneration, during normal aging, subtle changes not unlike those seen in the disease state could potentially contribute to a more gradual neuronal loss that could contribute to the cognitive decline seen in the aging population. This review provides illustrations of what is known about the role of microglia in neurodegeneration and makes suggestions about the role of microglia in age-related changes to the brain.

Early dysfunction of central 5-HT system in a murine model of bovine spongiform encephalopathy.

The hypothesis of an early vulnerability of the serotonergic system to prion infection was investigated in a murine model of bovine spongiform encephalopathy (BSE). Behavioral tests targeted to 5-HT functions were performed in the course of infection to evaluate circadian activity, anxiety-like behavior, pain sensitivity and the 5-HT syndrome. The first behavioral change was a decrease in nocturnal activity detected at 30% of incubation time. Further behavioral alterations including nocturnal hyperactivity, reduced anxiety, hyperalgesia and exaggerated 5-HT syndrome were observed at 60%-70% of incubation time, before the onset of clinical signs. The same tests performed in 5-HT-depleted mice and in prion protein-deficient mice revealed behavioral abnormalities similar in many aspects to those of BSE-infected mice. Histological and biochemical analysis showed alterations of the serotonergic system in BSE-infected and prion protein-deficient mice. These results indicate that BSE infection affects the homeostasis of serotonergic neurons and suggest that the disruption of prion protein normal function contributes to the early pathological changes in our mouse model of BSE. A similar process may occur in the human variant Creutzfeldt-Jacob disease, as suggested by the early symptoms of alterations in mood, sleep and pain sensitivity.

Mechanisms of prion protein aggregation.

The prion protein is a cell surface glycoprotein that is converted to a protease resistant abnormal isoform during the course of prion disease. The normal isoform of this protein has been shown to be an antioxidant that aids the survival of neurones. The abnormal isoform is associated with both the transmissible agent of prion diseases and is also toxic. Recent studies have shown that there are multiple end states in terms of aggregation of the protein. Both soluble oligomers and insoluble fibrils can form from the abnormal isoform. Although fibrils are characteristic of the disease, the most infectious prions are associated with oligomers. Neurotoxicity can be associated with fibrils but mostly appears to be due to small aggregates. For many years fibrils were believed to be central to the disease process but currently evidence supports the notion that fibrils represent a "bulk" form of abnormal protein, which is largely inert, but carried along a small active component. This review will examine what is known about the mechanisms behind prion protein aggregation, and the relevance of each form for the disease.

Prion interference with multiple prion isolates.

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.

Differential effects of prion particle size on infectivity in vivo and in vitro.

The conversion of cellular prion protein to the disease-associated isoform (PrP(Sc)) has been suggested to follow a mechanism of seeded aggregation. Here, we show that fragmentation of PrP(Sc) aggregates by sonication increases converting activity in cell culture in a way similar to in vitro conversion assays. In contrast, under the same conditions the infectious titer of sonicated samples in vivo was reduced. We modified the size distribution of PrP(Sc) by adsorption to nitrocellulose, which resulted in a reduction of the infectious titer in non-sonicated samples and an increase in sonicated samples. Our results indicate that NC-adsorption can (i) block some active sites of PrP(Sc) aggregates and (ii) reduce the rate of clearance from the brain. For large particles with low clearance the effect of NC-particles on the number of available active sites may dominate, whereas for smaller particles (i.e. sonicated samples) the effect of NC-adsorption on clearance dominates resulting in increased infectivity.

Overactivation of calcineurin induced by amyloid-beta and prion proteins.

Amyloid-beta protein (A beta) and the scrapie isoform of prion protein (PrPSs) have a central role in the pathogenesis of Alzheimer's disease (AD) and prion-related encephalopathies (PRE), respectively. In both disorders, the deposition of these misfolded proteins is accompanied by apoptotic neuronal loss. However, the pathogenesis and molecular basis of A beta- and PrPSc-neurotoxic effects are not completely understood. The Ca2+/calmodulin-dependent phosphatase calcineurin (CaN), through the dephosphorylation of the proapoptotic protein BAD, may be the link between Ca2+homeostasis deregulation and apoptotic neuronal death. In this study we used primary cultures of rat brain cortical neurons in order to investigate whether A beta and PrP affect CaN activity. We observed that synthetic peptides of A beta (A beta 25-35 and A beta 1-40) and PrP (PrP106-126) increased CaN activity, but did not affect the levels of this protein phosphatase. Moreover, we found that these peptides reduced the levels of BAD phosphorylated at serine residue 112, and this effect was prevented by the CaN inhibitor FK506. Since dephosphorylated BAD translocates to mitochondria, where it triggers cytochrome c release, we determined the levels of BAD in mitochondrial and cytosolic fractions. The data obtained showed that A beta- and PrP-treated neurons had higher levels of BAD in mitochondria than control neurons. This increase in mitochondrial BAD levels was matched by a decrease in cytochrome c. FK506 prevented the alterations of mitochondrial BAD and cytochrome c levels induced by A beta and PrP peptides. Taken together the data suggest that A beta and PrP increased CaN activity, inducing BAD dephosphorylation and translocation to mitochondria and, subsequently, cytochrome c release that may trigger an apoptotic cascade. Therefore, therapeutic strategies targeting CaN might be valuable for these neurodegenerative disorders.

Fatal neurological disease in scrapie-infected mice induced for experimental autoimmune encephalomyelitis.

During the years or decades of prion disease incubation, at-risk individuals are certain to encounter diverse pathological insults, such as viral and bacterial infections, autoimmune diseases, or inflammatory processes. Whether prion disease incubation time and clinical signs or otherwise the pathology of intercurrent diseases can be affected by the coinfection process is unknown. To investigate this possibility, mice infected with the scrapie agent at both high and low titers were subsequently induced for experimental autoimmune encephalomyelitis, an immune system-mediated model of central nervous system (CNS) inflammation. We show here that co-induced mice died from a progressive neurological disease long before control mice succumbed to classical scrapie. To investigate the mechanism of the co-induced syndrome, we evaluated biochemical and pathological markers of both diseases. Brain and spleen PrP(Sc) levels in the dying co-induced mice were comparable to those observed in asymptomatic scrapie-infected animals, suggesting that co-induced disease is not an accelerated form of scrapie. In contrast, inflammatory markers, such as demyelination, immune cell infiltrates, and gliosis, were markedly increased in co-induced mouse spinal cords. Activated astrocytes were especially elevated in the medulla oblongata. Furthermore, PrP(sc) depositions were found in demyelinated white matter areas in co-induced mouse spinal cords, suggesting the presence of activated infected immune cells that infiltrate into the CNS to facilitate the process of prion neuroinvasion. We hypothesize that inflammatory processes affecting the CNS may have severe clinical implications in subjects incubating prion diseases.

Insights into prion strains and neurotoxicity.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that are caused by prions and affect humans and many animal species. It is now widely accepted that the infectious agent that causes TSEs is PrP(Sc), an aggregated moiety of the host-derived membrane glycolipoprotein PrP(C). Although PrP(C) is encoded by the host genome, prions themselves encipher many phenotypic TSE variants, known as prion strains. Prion strains are TSE isolates that, after inoculation into distinct hosts, cause disease with consistent characteristics, such as incubation period, distinct patterns of PrP(Sc) distribution and spongiosis and relative severity of the spongiform changes in the brain. The existence of such strains poses a fascinating challenge to prion research.