Pα-syn* mitotoxicity is linked to MAPK activation and involves tau phosphorylation and aggregation at the mitochondria.

We recently identified a truncated and phosphorylated form of α-synuclein, pα-syn*, as a key neurotoxic α-synuclein species found in cultured neurons, as well as in mouse and Parkinson's disease patients' brains. Small pα-syn* aggregates localize to mitochondria and induce mitochondrial damage and fragmentation. Herein, we investigated the molecular basis of pα-syn*-induced toxicity. By immunofluorescence, we found phosphorylated MKK4, JNK, ERK5 and p38 MAPKs in pα-syn* inclusions. pJNK colocalized with pα-syn* at mitochondria and mitochondria-associated ER membranes where it was associated with BiP and pACC1, markers for the ER and energy deprivation, respectively. We also found that pα-syn* aggregates are tightly associated with small ptau aggregates of similar size. Pα-syn*/ptau inclusions localized to areas of mitochondrial damage and to mitophagic vesicles, showing their role in mitochondrial toxicity, mitophagy induction and their removal along with damaged mitochondrial fragments. Several MAPKs may act cooperatively to phosphorylate tau, notably JNK, p38 and GSK3ß, a non-MAPK that was also found phosphorylated in the vicinity of pα-syn*/ptau aggregates. These results add insight into the mechanisms by which pα-syn* exerts its toxic effects that include the phosphorylation of several kinases of the MAPK pathway, as well as the formation of ptau at the mitochondrial membrane, likely contributing to mitotoxicity. Thus pα-syn* appears to be the trigger of a series of kinase mediated pathogenic events and a link between α-syn pathology and tau, another protein known to aggregate in Parkinson's disease and other synucleinopathies.

Identifying therapeutic targets and treatments in model systems.

In this chapter, we describe current therapeutic targets for prion diseases. We focus on targets that have been validated in vitro and in vivo, leaving out a plethora of theoretic targets that still require validation. We also show how the development of improved model systems for the study of prion infection and neurotoxic mechanisms has enabled target identification. Some therapeutic targets are prion-specific, such as PrPTSE, while others are shared by other neurodegenerative diseases, for example, autophagy, cholesterol and energy metabolism, and neuroinflammation. In vivo models are discussed, and a genetic model of Creutzfeldt-Jakob disease particularly well suited for evaluation of prophylactic intervention is described in more detail.

Identification of a highly neurotoxic α-synuclein species inducing mitochondrial damage and mitophagy in Parkinson's disease.

Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.

Modeling Variant Creutzfeldt-Jakob Disease and Its Pathogenesis in Non-human Primates.

In the early 90s', Europe was shaken by the fear that the prions from "mad cow disease" (bovine spongiform encephalopathy) would transmit the disease to humans via beef products. In 1996, the first variant Creutzfeldt-Jakob (vCJD) patients were described, and the same year our Bovine Spongiform Encephalopathy (BSE) transmission studies to cynomolgus macaques demonstrated that the BSE prion was highly infectious for primates, inducing brain lesions identical to those observed in vCJD patients. These studies provided the first experimental evidence that vCJD was BSE in humans. Subsequent studies established the BSE/vCJD-infected cynomolgus macaque as a robust model to study the pathogenesis of vCJD. We showed rapid adaptation of BSE prions to primates upon subsequent passage, and their distribution in peripheral tissues and blood. Some key studies are summarized in the present paper.

Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment.

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD(+)) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD(+) followed by decreased ATP production, and are completely rescued by treatment with NAD(+) or its precursor nicotinamide because of restoration of physiological NAD(+) levels. Toxic prion protein-induced NAD(+) depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD(+). Intranasal NAD(+) treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD(+) starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD(+) replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.

Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis.

The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis.

Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP-FRET-enabled high throughput assay (PrP-FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood-brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.

Strain-specific role of RNAs in prion replication.

Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP(C) into PrP(Sc) in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP(Sc)-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.

Highly neurotoxic monomeric α-helical prion protein.

Prion diseases are infectious and belong to the group of protein misfolding neurodegenerative diseases. In these diseases, neuronal dysfunction and death are caused by the neuronal toxicity of a particular misfolded form of their cognate protein. The ability to specifically target the toxic protein conformer or the neuronal death pathway would provide powerful therapeutic approaches to these diseases. The neurotoxic forms of the prion protein (PrP) have yet to be defined but there is evidence suggesting that at least some of them differ from infectious PrP (PrP(Sc)). Herein, without making an assumption about size or conformation, we searched for toxic forms of recombinant PrP after dilution refolding, size fractionation, and systematic biological testing of all fractions. We found that the PrP species most neurotoxic in vitro and in vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form of PrP. TPrP caused autophagy, apoptosis, and a molecular signature remarkably similar to that observed in the brains of prion-infected animals. Interestingly, highly α-helical intermediates have been described for other amyloidogenic proteins but their biological significance remains to be established. We provide unique experimental evidence that a monomeric α-helical form of an amyloidogenic protein represents a cytotoxic species. Although toxic PrP has yet to be purified from prion-infected brains, TPrP might be the equivalent of one highly neurotoxic PrP species generated during prion replication. Because TPrP is a misfolded, highly neurotoxic form of PrP reproducing several features of prion-induced neuronal death, it constitutes a useful model to study PrP-induced neurodegenerative mechanisms.

Assessing prion infectivity of human urine in sporadic Creutzfeldt-Jakob disease.

Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP(Sc)). PrP(Sc) propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP(Sc) presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.

Prion strain discrimination based on rapid in vivo amplification and analysis by the cell panel assay.

Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.

Atypical BSE (BASE) transmitted from asymptomatic aging cattle to a primate.

BACKGROUND: Human variant Creutzfeldt-Jakob Disease (vCJD) results from foodborne transmission of prions from slaughtered cattle with classical Bovine Spongiform Encephalopathy (cBSE). Atypical forms of BSE, which remain mostly asymptomatic in aging cattle, were recently identified at slaughterhouses throughout Europe and North America, raising a question about human susceptibility to these new prion strains. METHODOLOGY/PRINCIPAL FINDINGS: Brain homogenates from cattle with classical BSE and atypical (BASE) infections were inoculated intracerebrally into cynomolgus monkeys (Macacca fascicularis), a non-human primate model previously demonstrated to be susceptible to the original strain of cBSE. The resulting diseases were compared in terms of clinical signs, histology and biochemistry of the abnormal prion protein (PrPres). The single monkey infected with BASE had a shorter survival, and a different clinical evolution, histopathology, and prion protein (PrPres) pattern than was observed for either classical BSE or vCJD-inoculated animals. Also, the biochemical signature of PrPres in the BASE-inoculated animal was found to have a higher proteinase K sensitivity of the octa-repeat region. We found the same biochemical signature in three of four human patients with sporadic CJD and an MM type 2 PrP genotype who lived in the same country as the infected bovine. CONCLUSION/SIGNIFICANCE: Our results point to a possibly higher degree of pathogenicity of BASE than classical BSE in primates and also raise a question about a possible link to one uncommon subset of cases of apparently sporadic CJD. Thus, despite the waning epidemic of classical BSE, the occurrence of atypical strains should temper the urge to relax measures currently in place to protect public health from accidental contamination by BSE-contaminated products.

In vitro and in vivo neurotoxicity of prion protein oligomers.

The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.

Toxic effects of intracerebral PrP antibody administration during the course of BSE infection in mice.

The absence of specific immune response is a hallmark of prion diseases. However, in vitro and in vivo experiments have provided evidence that an anti-PrP humoral response could have beneficial effects. Prophylactic passive immunization performed at the time of infection delayed or prevented disease. Nonetheless, the potential therapeutic effect of PrP antibodies administered shortly before the clinical signs has never been tested in vivo. Moreover, a recent study showed the potential toxicity of PrP antibodies administered intracerebrally. We aimed at evaluating the effect of a prolonged intracerebral anti-PrP antibody administration at the time of neuroinvasion in BSE infected Tg20 mice. Unexpectedly, despite a good penetration of the antibodies in the brain parenchyma, the treatment was not protective against the development of BSE. Instead, it led to an extensive neuronal loss, strong astrogliosis and microglial activation. Since this effect was observed after injection of anti-PrP antibodies as whole IgGs, F(ab')(2) or Fab fragments, the toxicity was directly related to the ability of the antibodies to recognize native PrP and to the intracerebral concentration achieved, and not to the Fc portion or the divalence of the antibodies. This experiment shows that a prolonged treatment with anti-PrP antibodies by the intracerebral route can induce severe side-effects and calls for caution with regard to the use of similar approaches for late therapeutic interventions in humans.

The 37-kDa/67-kDa laminin receptor acts as a receptor for infectious prions and is inhibited by polysulfated glycanes.

BACKGROUND: Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS: For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS: The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS: We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.

Preclinical metabolic changes in mouse prion diseases detected by 1H-nuclear magnetic resonance spectroscopy.

Magnetic resonance spectroscopy studies in animal models of prion disease are very few and concern terminal stages of infection. In order to study earlier stages of the disease, we used in-vivo magnetic resonance spectroscopy in a mouse model of scrapie and, for the first time, in mice infected with a bovine spongiform encephalopathy strain. In bovine spongiform encephalopathy-infected mice, we observed an increase in myo-inositol preceding clinical signs by 20 days, followed by a decrease in N-acetylaspartate at advanced stages. In scrapie-infected mice, changes in N-acetylaspartate and myo-inositol were detected at the beginning of the symptomatic phase. These results show that magnetic resonance spectroscopy is a valuable tool for detecting subtle metabolic changes associated to gliosis and neuronal dysfunction in prion diseases.

PrPTSE distribution in a primate model of variant, sporadic, and iatrogenic Creutzfeldt-Jakob disease.

Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), are neurodegenerative and fatal. Sporadic CJD (sCJD) can be transmitted between humans through medical procedures involving highly infected organs, such as the central nervous system. However, in variant CJD (vCJD), which is due to human contamination with the bovine spongiform encephalopathy (BSE) agent, lymphoreticular tissue also harbors the transmissible spongiform encephalopathy-associated prion protein (PrP(TSE)), which poses a particularly acute risk for iatrogenic transmission. Two blood transfusion-related cases are already documented. In addition, the recent observation of PrP(TSE) in spleen and muscle in sCJD raised the possibility that peripheral PrP(TSE) is not limited to vCJD cases. We aimed to clarify the peripheral pathogenesis of human TSEs by using a nonhuman primate model which mimics human diseases. A highly sensitive enzyme-linked immunosorbent assay was adapted to the detection of extraneural PrP(TSE). We show that affected organs can be divided into two groups. The first is peripheral organs accumulating large amounts of PrP(TSE), which represent a high risk of iatrogenic transmission. This category comprises only lymphoreticular organs in the vCJD/BSE model. The second is organs with small amounts of PrP(TSE) associated with nervous structures. These are the muscles, adrenal glands, and enteric nervous system in the sporadic, iatrogenic, and variant CJD models. In contrast to the first set of organs, this low level of tissue contamination is not strain restricted and seems to be linked to secondary centrifugal spread of the agent through nerves. It might represent a risk for iatrogenic transmission, formerly underestimated despite previous reports of low rates of transmission from peripheral organs of humans to nonhuman primates (5, 10). This study provides an additional experimental basis for the classification of human organs into different risk categories and a rational re-evaluation of current risk management measures.