In vivo and in vitro characterization of an infectious synthetic prion amyloid

Prion pathologies are a group of fatal neurodegenerative disorders that afflict mammalian species. In these diseases the cellular prion protein (PrPC) misfolds into a disease related form, known as prion (PrPSc). This misfolded protein is able to recruit and convert the physiological PrPC into pathogenic PrPSc that accumulates and leads to neuronal death. Synthetic infectious prions generated from recombinant source of PrP (recPrP) were created in the past few years with the aim of assessing if these preparations could mimic the behaviors of natural prions. Particularly, synthetic prions offer a great tool for studying both the basis of prion replication and the mechanism behind the evolution of prion strains. Indeed, recent evidence suggests that, despite the absence of nucleic acids, prions can adapt their conformation in response to changes in the context of replication. In this thesis, we have analyzed the stability of a synthetic prion isolate previously generated and named amyloid #4. In particular, amyloid #4 was either injected in wild-type mice or amplified by means of an innovative technique named Protein Misfolding Cyclic Amplfication (PMCA) which is able to mimic in vitro the process of prion conversion which occurs in vivo. Injection of raw amyloid failed to induce any disease while the injection of the PMCA amplified material induced severe prion pathology characterized by the presence of an uncommon PrPSc. When further passaged in wild-type animals such PrPSc was able to differentiate in two different isolates able to sustain different types of prion disease. These two isolates were finally assessed in PMCA and we observed the generation of an alternative conformation whose biophysical properties were different from those of both inocula, suggesting that the in vitro context of replication selectively amplified a third PrPSc isolate. Taken together these results indicate that our synthetic prion undergoes to process of evolution (selection/adaptation) dictated by the environment of its replication (bioassays VS PMCA). Synthetic prions can be used for studying prion adaptation and selection. Since all the proposed pharmacological treatments failed to cure these pathologies, understanding these behaviors of prions is of fundamental importance to design effective therapies for these devastating disorders

Detection of prion seeding activity in the olfactory mucosa of patients with Fatal Familial Insomnia

Fatal Familial Insomnia (FFI) is a genetic prion disease caused by a point mutation in the prion protein gene (PRNP) characterized by prominent thalamic atrophy, diffuse astrogliosis and moderate deposition of PrP Sc in the brain. Here, for the first time, we demonstrate that the olfactory mucosa (OM) of patients with FFI contains trace amount of PrP Sc detectable by PMCA and RT-QuIC. Quantitative PMCA analysis estimated a PrP Sc concentration of about 1 \uc3\u97 10-14g/ml. In contrast, PrP Sc was not detected in OM samples from healthy controls and patients affected by other neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and frontotemporal dementia. These results indicate that the detection limit of these assays is in the order of a single PrP Sc oligomer/molecule with a specificity of 100%

PMCA-based detection of prions in the olfactory mucosa of patients with Sporadic Creutzfeldt-Jakob Disease

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials

Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP(Sc). Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP(Sc) were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties

Contributions of Molecular and Optical Techniques to the Clinical Diagnosis of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide. The distinctive neuropathological feature of AD is the intracerebral accumulation of two abnormally folded proteins: β-amyloid (Aβ) in the form of extracellular plaques, and tau in the form of intracellular neurofibrillary tangles. These proteins are considered disease-specific biomarkers, and the definite diagnosis of AD relies on their post-mortem identification in the brain. The clinical diagnosis of AD is challenging, especially in the early stages. The disease is highly heterogeneous in terms of clinical presentation and neuropathological features. This phenotypic variability seems to be partially due to the presence of distinct Aβ conformers, referred to as strains. With the development of an innovative technique named Real-Time Quaking-Induced Conversion (RT-QuIC), traces of Aβ strains were found in the cerebrospinal fluid of AD patients. Emerging evidence suggests that different conformers may transmit their strain signature to the RT-QuIC reaction products. In this review, we describe the current challenges for the clinical diagnosis of AD and describe how the RT-QuIC products could be analyzed by a surface-enhanced Raman spectroscopy (SERS)-based systems to reveal the presence of strain signatures, eventually leading to early diagnosis of AD with the recognition of individual disease phenotype

Assessment of doxycycline treatment on prion deposition in the olfactory epithelium of patients with Fatal Familial Insomnia: Possible mirroring of the CNS alterations

Fatal Familial Insomnia (FFI) is an autosomal dominant inherited prion disease due to a point mutation at position 178 of the prion protein gene (PRNP) resulting in aspartic acid to asparagine substitution (D178N) in coupling phase with methionine at position 129. The overall amount of protease resistant prion protein (PrPres) in FFI is 5-10 times lower than that found in classical forms of sporadic CJD and prevalently affects the thalamus. Recent data showed that prions can be found in the olfactory mucosa (OM) of patients with CJD (Zanusso et al. 2014). The aim of the present study was to verify whether PrPres is also present in the OM of FFI patients and, if this were the case, whether this marker can be used to evaluate the response to pharmacological treatment. First, we optimized the protein misfolding cyclic amplification (PMCA) on samples of frontal cortex from FFI patients. The optimized method allowed to detect a PrPres signal till 10-12-fold dilution of brain homogenate. We then analyzed the OM from a symptomatic FFI patient and found a PrPres signal with a biochemical profile similar to that found in FFI brain. Quantitative PMCA showed that the PrPres concentration in the OM was approximately 1.4 × 10-18 grams/10 microliters. We are currently extending the study to members of a large Italian kindred of FFI involved in a clinical trial aimed to assess the effect of doxycycline (administered at preclinical stage) on disease progression. OM samples, collected at both preclinical and clinical stage of doxycycline-treated and -untreated individuals, will be analyzed to assess whether PrPres can be detected at a pre-symptomatic stage and if the treatment reduces the amount of PrPres or alters its biochemical properties. Since OM belongs to the CNS, the analysis of OM-PrPres might mirror the modifications occurring in the CNS, thus providing important information about the real effect of the doxycycline on prion propagation

Animal Models of Autosomal Recessive Parkinsonism

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. The neuropathological hallmark of the disease is the loss of dopamine neurons of the substantia nigra pars compacta. The clinical manifestations of PD are bradykinesia, rigidity, resting tremors and postural instability. PD patients often display non-motor symptoms such as depression, anxiety, weakness, sleep disturbances and cognitive disorders. Although, in 90% of cases, PD has a sporadic onset of unknown etiology, highly penetrant rare genetic mutations in many genes have been linked with typical familial PD. Understanding the mechanisms behind the DA neuron death in these Mendelian forms may help to illuminate the pathogenesis of DA neuron degeneration in the more common forms of PD. A key step in the identification of the molecular pathways underlying DA neuron death, and in the development of therapeutic strategies, is the creation and characterization of animal models that faithfully recapitulate the human disease. In this review, we outline the current status of PD modeling using mouse, rat and non-mammalian models, focusing on animal models for autosomal recessive PD

Neurotoxicity and synaptic plasticity impairment of N-acetylglucosamine polymers: implications for Alzheimer’s disease

Here we assessed whether polymers of GlcNAc have any pathogenetic role in AD. First, by using specific dyes we found deposits of polymers of GlcNAc in sporadic, but not in familial AD. We found that neurons and microglia exposed to GlcNAc and UDP-GlcNAc are able to form GlcNAc polymers, which display a significant neurotoxicity in vitro. Moreover, the exposure of organotypic hippocampal cultures to the same compounds led to synaptic impairment with decreased levels of syntaxin and synaptophysin. In addition, acute hippocampal slices treated with GlcNAc/UDP-GlcNAc showed a clear reduction of long-term potentiation of excitatory synapses. Finally, we demonstrated that microglial cells are able to phagocytose chitin particles and, when exposed to GlcNAc/UDP-GlcNAc, show cellular activation and intracellular deposition of GlcNAc polymers which are eventually released in the extracellular space. Taken together, our results indicate that both microglia and neurons produce GlcNAc polymers, which trigger neurotoxicity both directly and through microglia activation. GlcNAc polymer-driven neurotoxicity offers novel pathogenic insights in sporadic AD and new therapeutic options

Synthetic Prion Selection and Adaptation

Prion pathologies are characterized by the conformational conversion of the cellular prion protein (PrPC) into a pathological infectious isoform, known as PrPSc. The latter acquires different abnormal conformations, which are associated with specific pathological phenotypes. Recent evidence suggests that prions adapt their conformation to changes in the context of replication. This phenomenon is known as either prion selection or adaptation, where distinct conformations of PrPSc with higher propensity to propagate in the new environment prevail over the others. Here, we show that a synthetically generated prion isolate, previously subjected to protein misfolding cyclic amplification (PMCA) and then injected in animals, is able to change its biochemical and biophysical properties according to the context of replication. In particular, in second transmission passage in vivo, two different prion isolates were found: one characterized by a predominance of the monoglycosylated band (PrPSc-M) and the other characterized by a predominance of the diglycosylated one (PrPSc-D). Neuropathological, biochemical, and biophysical assays confirmed that these PrPSc possess distinctive characteristics. Finally, PMCA analysis of PrPSc-M and PrPSc-D generated PrPSc (PrPSc-PMCA) whose biophysical properties were different from those of both inocula, suggesting that PMCA selectively amplified a third PrPSc isolate. Taken together, these results indicate that the context of replication plays a pivotal role in either prion selection or adaptation. By exploiting the ability of PMCA to mimic the process of prion replication in vitro, it might be possible to assess how changes in the replication environment influence the phenomenon of prion selection and adaptation

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1+/+), hemizygous (Pin1+/−) or knock-out (Pin1−/−) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease