Search

Semantic and right temporal variant of frontotemporal dementia (svFTD and rtvFTD) are rare clinical phenotypes in which, in most cases, the underlying pathology is TDP-43 proteinopathy. They are usually sporadic disorders, but recent evidences suggest a higher frequency of genetic mutations for the right temporal versus the semantic variant. However, the genetic basis of these forms is not clear. In this study we performed a genetic screening of a single-center cohort of svFTD and rtvFTD patients, aiming at identifying the associated genetic variants. A panel of 73 dementia candidate genes has been analyzed by NGS target sequencing including both causal and risk/modifier genes in 23 patients (15 svFTD and 8 rtvFTD) and 73 healthy age-matched controls. We first performed a single variant analysis considering rare variants and then a gene-based aggregation analysis to evaluate the cumulative effects of multiple rare variants in a single gene. We found 12 variants in nearly 40% of patients (9/23), described as pathogenic or classified as VUS/likely pathogenic. The overall rate was higher in svFTD than in rtvFTD. Three mutations were located in MAPT gene and single mutations in the following genes: SQSTM1, VCP, PSEN1, TBK1, OPTN, CHCHD10, PRKN, DCTN1 . Our study revealed the presence of variants in genes involved in pathways relevant for the pathology, especially autophagy and inflammation. We suggest that molecular analysis should be performed in all svFTD and rtvFTD patients, to better understand the genotype-phenotype correlation and the pathogenetic mechanisms that could drive the clinical phenotypes in FTD., Competing Interests: EC has received research supports from the Italian Ministry of Health. MF is Editor-in-Chief of the Journal of Neurology; received compensation for consulting services and/or speaking activities from Bayer, Biogen Idec, Merck-Serono, Novartis, Roche, Sanofi Genzyme, Takeda, and Teva Pharmaceutical Industries; and receives research support from Biogen Idec, Merck-Serono, Novartis, Roche, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, and ARiSLA (Fondazione Italiana di Ricerca per la SLA). FA is Section Editor of NeuroImage: Clinical; has received speaker honoraria from Biogen Idec, Roche and Zambon; and receives or has received research supports from the Italian Ministry of Health, AriSLA (Fondazione Italiana di Ricerca per la SLA), the European Research Council and the Foundation Research on Alzheimer Disease. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rossi, Salvi, Mehmeti, Ricci, Villa, Prioni, Moda, Di Fede, Tiraboschi, Redaelli, Coppola, Koch, Canu, Filippi, Agosta, Giaccone and Caroppo.)

Parkinson's disease (PD) is a neurodegenerative disorder often associated with pre-motor symptoms involving both gastrointestinal and olfactory tissues. PD patients frequently suffer from hyposmia, hyposalivation, dysphagia and gastrointestinal dysfunctions. During the last few years it has been speculated that microbial agents could play a crucial role in PD. In particular, alterations of the microbiota composition (dysbiosis) might contribute to the formation of misfolded α-synuclein, which is believed to be the leading cause of PD. However, while several findings confirmed that there might be an important link between intestinal microbiota alterations and PD onset, little is known about the potential contribution of the nasal microbiota. Here, we describe the latest findings on this topic by considering that more than 80% of patients with PD develop remarkable olfactory deficits in their prodromal disease stage. Therefore, the nasal microbiota might contribute to PD, eventually boosting the gut microbiota in promoting disease onset. Finally, we present the applications of the seed amplification assays to the study of the gut and olfactory mucosa of PD patients, and how they could be exploited to investigate whether pathogenic bacteria present in the gut and the nose might promote α-synuclein misfolding and aggregation.

The conformational conversion of the cellular prion protein (PrP C ) into a misfolded, aggregated and infectious scrapie isoform is associated with prion disease pathology and neurodegeneration. Despite the significant number of experimental and theoretical studies the molecular mechanism regulating this structural transition is still poorly understood. Here, via Nuclear Magnetic Resonance (NMR) methodologies we investigate at the atomic level the mechanism of the human HuPrP(90-231) thermal unfolding and characterize the conformational equilibrium between its native structure and a β-enriched intermediate state, named β-PrPI. By comparing the folding mechanisms of metal-free and Cu 2+ -bound HuPrP(23-231) and HuPrP(90-231) we show that the coupling between the N- and C-terminal domains, through transient electrostatic interactions, is the key molecular process in tuning long-range correlated μs-ms dynamics that in turn modulate the folding process. Moreover, via thioflavin T (ThT)-fluorescence fibrillization assays we show that β-PrPI is involved in the initial stages of PrP fibrillation, overall providing a clear molecular description of the initial phases of prion misfolding. Finally, we show by using Real-Time Quaking-Induced Conversion (RT-QuIC) that the β-PrPI acts as a seed for the formation of amyloid aggregates with a seeding activity comparable to that of human infectious prions., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Parkinson disease (PD) is the second most common neurodegenerative disease, and the most common synucleinopathy, as alpha-synuclein (α-syn), a prion-like protein, plays an important pathophysiologic role in its onset and progression. Although neuropathologic changes begin many years before the onset of motor manifestations, diagnosis still relies on the identification of the motor symptoms, which hinders to formulate an early diagnosis. Because α-syn misfolding and aggregation precede clinical manifestations, the possibility to identify these phenomena in patients with PD would allow us to recognize the disease at the earliest, premotor phases, as a consequence of the transition from a clinical to a molecular diagnosis. Seed amplification assays (SAAs) are a group of techniques that currently support the diagnosis of prion subacute encephalopathies, namely Creutzfeldt-Jakob disease. These techniques enable the detection of minimal amounts of prions in CSF and other matrices of affected patients. Recently, SAAs have been successfully applied to detect misfolded alpha-synuclein (α-syn) in CSF, olfactory mucosa, submandibular gland biopsies, skin, and saliva of patients with Parkinson disease (PD) and other synucleinopathies. In these categories, they can differentiate PD and dementia with Lewy bodies (DLBs) from control subjects, even in the prodromal stages of the disease. In differential diagnosis, SAAs satisfactorily differentiated PD, DLB, and multiple system atrophy (MSA) from nonsynucleinopathy parkinsonisms. The kinetic analysis of the SAA fluorescence profiles allowed the identification of synucleinopathy-dependent α-syn fibrils conformations, commonly referred to as strains, which have demonstrated diagnostic potential in differentiating among synucleinopathies, especially between Lewy body diseases (LBDs) (PD and DLB) and MSA. In front of these highly promising data, which make the α-syn seeding activity detected by SAAs as the most promising diagnostic biomarker for synucleinopathies, there are still preanalytical and analytical issues, mostly related to the assay standardization, which need to be solved. In this review, we discuss the key findings supporting the clinical application of α-syn SAAs to identify PD and other synucleinopathies, the unmet needs, and future perspectives., (© 2022 American Academy of Neurology.)

Background and Purpose: Mutations in DNAJB2 are associated with autosomal recessive hereditary motor neuropathies/ Charcot-Marie-Tooth disease type 2 (CMT2). We describe an Italian family with CMT2 due to a homozygous DNAJB2 mutation and provide insight into the pathomechanisms., Methods: Patients with DNAJB2 mutations were characterized clinically, electrophysiologically and by means of skin biopsy. mRNA and protein levels were studied in lymphoblastoid cells (LCLs) from patients and controls., Results: Three affected siblings were found to carry a homozygous DNAJB2 null mutation segregating with the disease. The disease manifested in the second to third decade of life. Clinical examination showed severe weakness of the thigh muscles and complete loss of movement in the foot and leg muscles. Sensation was reduced in the lower limbs. All patients had severe hearing loss and the proband also had Parkinson's disease (PD). Nerve conduction studies showed an axonal motor and sensory length-dependent polyneuropathy. DNAJB2 expression studies revealed reduced mRNA levels and the absence of the protein in the homozygous subject in both LCLs and skin biopsy. Interestingly, we detected phospho-alpha-synuclein deposits in the proband, as already seen in PD patients, and demonstrated TDP-43 accumulation in patients' skin., Conclusions: Our results broaden the clinical spectrum of DNAJB2-related neuropathies and provide evidence that DNAJB2 mutations should be taken into account as another causative gene of CMT2 with hearing loss and parkinsonism. The mutation likely acts through a loss-of-function mechanism, leading to toxic protein aggregation such as TDP-43. The associated parkinsonism resembles the classic PD form with the addition of abnormal accumulation of phospho-alpha-synuclein., (© 2022 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Background: Rapidly progressive cognitive impairment is a diagnostic criterion in Creutzfeldt-Jakob disease (CJD), but the diagnosis is usually reached when an analysis of cognitive aspects is no longer possible., Objective: This study aims to delineate the cognitive phenotypes preceding severe dementia in CJD compared to secondary metabolic encephalopathies (SME) with rapid cognitive impairment., Methods: Patients with rapidly progressive neurological symptoms underwent neuropsychological evaluation, analysis of cerebrospinal fluid (CSF) and codon 129 polymorphism of the prion protein gene (PRNP), magnetic resonance imaging (MRI), and single positron emission computed tomography ( 99mTc SPECT). CSF real-time quaking-induced conversion analysis was applied in CJD patients. Based on literature and clinical expertise, cognitive profiles were correlated with brain areas., Results: Thirty-one patients were diagnosed with CJD (n = 17) or SME; 77 cases of CJD were extracted from the literature. In patients with CJD, verbal initiative, lexical search, long-term memory, attention, and abstract reasoning were the most frequently impaired abilities. Cognitive profiles were mainly related to dysfunction in fronto-temporal areas. Furthermore, they were consistent with areas of hypoperfusion detected by 99mTc SPECT in six patients and cortical and subcortical MRI hyperintensities in eight and 14 patients, respectively, and were similar to those described in the literature. In contrast, cognitive profiles were different from those in SME characterized by visuospatial and constructive deficits relating to posterior brain areas., Conclusion: In CJD, clinical and neuropsychological analyses outline a salient cognitive phenotype suggestive of fronto-temporal dysfunction preceding severe dementia. This phenotype is different from that observed in other rapidly progressive encephalopathies., (© 2021. Fondazione Società Italiana di Neurologia.)

Serpins represent the most broadly distributed superfamily of proteases inhibitors. They contribute to a variety of physiological functions and any alteration of the serpin-protease equilibrium can lead to severe consequences. SERPINA3 dysregulation has been associated with Alzheimer's disease (AD) and prion diseases. In this study, we investigated the differential expression of serpin superfamily members in neurodegenerative diseases. SERPIN expression was analyzed in human frontal cortex samples from cases of sporadic Creutzfeldt-Jakob disease (sCJD), patients at early stages of AD-related pathology, and age-matched controls not affected by neurodegenerative disorders. In addition, we studied whether Serpin expression was dysregulated in two animal models of prion disease and AD.Our analysis revealed that, besides the already observed upregulation of SERPINA3 in patients with prion disease and AD, SERPINB1, SERPINB6, SERPING1, SERPINH1, and SERPINI1 were dysregulated in sCJD individuals compared to controls, while only SERPINB1 was upregulated in AD patients. Furthermore, we analyzed whether other serpin members were differentially expressed in prion-infected mice compared to controls and, together with SerpinA3n, SerpinF2 increased levels were observed. Interestingly, SerpinA3n transcript and protein were upregulated in a mouse model of AD. The SERPINA3/SerpinA3nincreased anti-protease activity found in post-mortem brain tissue of AD and prion disease samples suggest its involvement in the neurodegenerative processes. A SERPINA3/SerpinA3n role in neurodegenerative disease-related protein aggregation was further corroborated by in vitro SerpinA3n-dependent prion accumulation changes. Our results indicate SERPINA3/SerpinA3n is a potential therapeutic target for the treatment of prion and prion-like neurodegenerative diseases., (© 2022. The Author(s).)

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrP C ) into an abnormally folded form, named prion (or PrP Sc ). 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 PrP Sc type 1 or 2) gives rise to different PrP Sc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrP Sc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrP Sc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrP Sc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrP Sc 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 PrP C 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 PrP Sc 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 PrP Sc was generated, regardless of the tissues analyzed. Notably, all amplified PrP Sc 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cazzaniga, Bistaffa, De Luca, Portaleone, Catania, Redaelli, Tramacere, Bufano, Rossi, Caroppo, Giovagnoli, Tiraboschi, Di Fede, Eleopra, Devigili, Elia, Cilia, Fiorini, Bongianni, Salzano, Celauro, Quarta, Mammana, Legname, Tagliavini, Parchi, Zanusso, Giaccone and Moda.)

Importance: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rapidly lethal disease. Rapid, accurate diagnosis is imperative for epidemiological surveillance and public health activities to exclude treatable differentials and facilitate supportive care. In 2017, the International CJD Surveillance Network diagnostic criteria were revised to incorporate cortical ribboning on magnetic resonance imaging and the real-time quaking-induced conversion (RT-QuIC) assay, developments that require multicenter evaluation., Objective: To evaluate the accuracy of revised diagnostic criteria through the retrospective diagnosis of autopsy-confirmed cases (referred to as in-life diagnosis)., Design, Setting, and Participants: This diagnostic study used a 3-year clinicopathological series using all cases of autopsy-confirmed sCJD and a noncase group with alternative neuropathological diagnoses from national surveillance centers in the United Kingdom, France, Germany, and Italy. Data were collected from January 2017 to December 2019 and analyzed from January 2020 to November 2021., Main Outcomes and Measures: Sensitivity and specificity of revised diagnostic criteria and diagnostic investigations. Secondary analyses assessing sCJD subgroups by genotype, pathological classification, disease duration, and age., Results: A total of 501 sCJD cases and 146 noncases were included. Noncase diagnoses included neurodegenerative diseases, autoimmune encephalitis, and cerebral insults such as anoxia. Participants in the sCJD cases cohort were younger (mean [SD] age, 68.8 [9.8] years vs 72.8 [10.9] years; P < .001) and had longer median (IQR) disease duration (118 [74.8-222.3] days vs 85 [51.5-205.5] days; P = .002); sex ratios were equivalent (253 [50.5%] male cases vs 74 [50.7%] male noncases). Sensitivity of revised criteria in in-life diagnosis (450 of 488 [92.2%] diagnoses; 95% CI, 89.5%-94.4%) was increased compared with prior criteria (378 of 488 [77.5%] diagnoses; 95% CI, 73.5%-81.1%; P < .001), while specificity (101 of 125 [80.8%] diagnoses; 95% CI, 72.8%-87.3%) was unchanged (102 of 125 [81.6%] diagnoses; 95% CI, 73.7%-88.0%; P > .99). Among 223 cases and 52 noncases with the full panel of investigations performed, sensitivity of revised criteria (97.8%; 95% CI, 94.9%-99.3%) was increased compared with prior criteria (76.2%; 95% CI, 70.1%-81.7%; P < .001) while specificity was unchanged (67.3%; 95% CI, 52.9%-79.7% vs 69.2%; 95% CI, 54.9%-81.3%; P > .99). In 455 cases and 111 noncases, cortical ribboning was 67.9% sensitive (95% CI, 63.4%-72.2%) and 86.5% specific (95% CI, 78.7%-92.2%). In 274 cases and 77 noncases, RT-QuIC was 91.6% sensitive (95% CI, 87.7%-94.6%) and 100% specific (95% CI, 96.2%-100%). Investigation sensitivity varied with genetic and pathological features, disease duration, and age., Conclusions and Relevance: This diagnostic study demonstrated significantly improved sensitivity of revised sCJD diagnostic criteria with unaltered specificity. The revision has enhanced diagnostic accuracy for clinical care and surveillance.

Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSyn D ). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn&#95;RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn&#95;RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn&#95;RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSyn D strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn&#95;RT-QuIC products.

Background: Detection of the pathological and disease-associated alpha-synuclein (αSyn D ) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson's disease (PD). We recently showed that αSyn D can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn&#95;RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes., Methods: OM samples were prospectively collected from patients with a probable diagnosis of MSA-P (n = 20, mean disease duration 4.4 years), MSA-C (n = 10, mean disease duration 4 years), PD (n = 13, mean disease duration 8 years), and healthy control subjects (HS) (n = 11). Each sample was analyzed by αSyn&#95;RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn&#95;RT-QuIC analytical procedures. Results were correlated with demographic and clinical data., Results: The αSyn&#95;RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83-1.00). In particular, αSyn&#95;RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn&#95;RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn&#95;RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability., Conclusions: Our study provides evidence that OM-αSyn D may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn&#95;RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials., (© 2021. The Author(s).)

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare and fatal neurodegenerative disorder with an incidence of 1.5 to 2 cases per million population/year. The disease is caused by a proteinaceous infectious agent, named prion (or PrPSc), which arises from the conformational conversion of the cellular prion protein (PrPC). Once formed, PrPSc interacts with the normally folded PrPC coercing it to undergo similar structural rearrangement. The disease is highly heterogeneous from a clinical and neuropathological point of view. The origin of this variability lies in the aberrant structures acquired by PrPSc. At least six different sCJD phenotypes have been described and each of them is thought to be caused by a peculiar PrPSc strain. Definitive sCJD diagnosis requires brain analysis with the aim of identifying intracerebral accumulation of PrPSc which currently represents the only reliable biomarker of the disease. Clinical diagnosis of sCJD is very challenging and is based on the combination of several clinical, instrumental and laboratory tests representing surrogate disease biomarkers. Thanks to the advent of the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, PrPSc was found in several peripheral tissues of sCJD patients, sometimes even before the clinical onset of the disease. This discovery represents an important step forward for the clinical diagnosis of sCJD. In this manuscript, we present an overview of the current applications and future perspectives of RT-QuIC in the field of sCJD diagnosis.

Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrP C , has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrP C levels to investigate the link between PrP C expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrP C , PrP Sc , in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrP C , which encompass almost all pathological amyloids involved in neurodegeneration.

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (β2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m promotes β2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1β (IL-1β) and IL-18. This process depends on activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lack efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Our results provide mechanistic evidence for β2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM., Competing Interests: Declaration of interests H.B. received research support from Celgene and Morphosys. The remaining authors declare no completing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is the most common type of dementia in the elderly population. The disease is characterized by progressive memory loss, cerebral atrophy, extensive neuronal loss, synaptic alterations, brain inflammation, extracellular accumulation of amyloid-β (Aβ) plaques, and intracellular accumulation of hyper-phosphorylated tau (p-tau) protein. Many recent clinical trials have failed to show therapeutic benefit, likely because at the time in which patients exhibit clinical symptoms the brain is irreversibly damaged. In recent years, induced pluripotent stem cells (iPSCs) have been suggested as a promising cell therapy to recover brain functionality in neurodegenerative diseases such as AD. To evaluate the potential benefits of iPSCs on AD progression, we stereotaxically injected mouse iPSC-derived neural precursors (iPSC-NPCs) into the hippocampus of aged triple transgenic (3xTg-AD) mice harboring extensive pathological abnormalities typical of AD. Interestingly, iPSC-NPCs transplanted mice showed improved memory, synaptic plasticity, and reduced AD brain pathology, including a reduction of amyloid and tangles deposits. Our findings suggest that iPSC-NPCs might be a useful therapy that could produce benefit at the advanced clinical and pathological stages of AD.

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.

Background: Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA)., Methods: In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology., Results: All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate., Conclusions: Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious., Funding: This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer's Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02)., Competing Interests: EB, AM, JE, CD, FC, SP, LC, GL, GG, JT, FM No competing interests declared, (© 2021, Bistaffa et al.)

Tauopathies are prevalent, invariably fatal brain diseases for which no cure is available. Tauopathies progressively affect the brain through cell-to-cell transfer of tau protein amyloids, yet the spreading mechanisms remain unknown. Here we show that the cellular prion protein (PrP C ) facilitates the uptake of tau aggregates by cultured cells, possibly by acting as an endocytic receptor. In mouse neuroblastoma cells, pull-down experiments revealed that tau amyloids bind to PrP C . Confocal images of both wild-type and PrP C -knockout N2a cells treated with fluorescently labeled synthetic tau fibrils showed that the internalization was reduced in isogenic cells devoid of the gene encoding PrP C . Pre-treatment of the same cells with antibodies against N-proximal epitopes of PrP C impaired the binding of tau amyloids and decreased their uptake. Surprisingly, exposure of chronically prion-infected cells to tau amyloids reduced the accumulation of aggregated prion protein and this effect lasted for more than 72 hr after amyloid removal. These results point to bidirectional interactions between the two proteins: while PrP C mediates the entrance of tau fibrils in cells, PrP Sc buildup is greatly reduced in their presence, possibly because of an impairment in the prion conversion process., (© 2020 International Society for Neurochemistry.)

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.

The pathological deposition of the transactive response DNA-binding protein of 43 kDa occurs in the majority (∼97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments capable of interfering with the underlying pathological process and early detection of transactive response DNA-binding protein of 43 kDa pathology would facilitate the development of disease-modifying drugs. The real-time quaking-induced conversion reaction showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite transactive response DNA-binding protein of 43 kDa displays prion-like properties, to date the real-time quaking-induced conversion reaction technology has not yet been adapted to this protein. The aim of this study was to adapt the real-time quaking-induced conversion reaction technique for the transactive response DNA-binding protein of 43 kDa substrate and to exploit the intrinsic ability of this technology to amplify minute amount of mis-folded proteins for the detection of pathological transactive response DNA-binding protein of 43 kDa species in the cerebrospinal fluid of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic transactive response DNA-binding protein of 43 kDa-pre-formed aggregates and with autopsy-verified brain homogenate samples and subsequently analysed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. Transactive response DNA-binding protein of 43 kDa real-time quaking-induced conversion reaction was able to detect as little as 15 pg of transactive response DNA-binding protein of 43 kDa aggregates, discriminating between a cohort of patients affected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration and age-matched controls with a total sensitivity of 94% and a specificity of 85%. Our data give a proof-of-concept that transactive response DNA-binding protein of 43 kDa is a suitable substrate for the real-time quaking-induced conversion reaction. Transactive response DNA-binding protein of 43 kDa real-time quaking-induced conversion reaction could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The cerebrospinal fluid detection of transactive response DNA-binding protein of 43 kDa pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Prion diseases are neurodegenerative and invariably fatal conditions that affect humans and animals. In particular, Creutzfeldt-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE) are paradigmatic forms of human and animal prion diseases, respectively. Human exposure to BSE through contaminated food caused the appearance of the new variant form of CJD (vCJD). These diseases are caused by an abnormal prion protein named PrP Sc (or prion), which accumulates in the brain and leads to the onset of the disease. Their definite diagnosis can be formulated only at post-mortem after biochemical and neuropathological identification of PrP Sc . Thanks to the advent of an innovative technique named protein misfolding cyclic amplification (PMCA), traces of PrP Sc , undetectable with the standard diagnostic techniques, were found in peripheral tissues of patients with vCJD, even at preclinical stages. The technology is currently being used in specialized laboratories and can be exploited for helping physicians in formulating an early and definite diagnosis of vCJD using peripheral tissues. However, this assay is currently unable to detect prions associated with the sporadic CJD (sCJD) forms, which are more frequent than vCJD. This review will focus on the most recent advances and applications of PMCA in the field of vCJD and other human prion disease diagnosis., Competing Interests: The authors declare no conflict of interest

Here, we demonstrate that introduction of halogen atoms at the tyrosine 10 phenol ring of the DSGYEV sequence derived from the flexible amyloid-β N -terminus, promotes its self-assembly in the solid state. In particular, we report the crystal structures of two halogen-modified sequences, which we found to be stabilized in the solid state by halogen-mediated interactions. The structural study is corroborated by Non-Covalent Interaction (NCI) analysis. Our results prove that selective halogenation of an amino acid enhances the supramolecular organization of otherwise unstructured biologically-relevant sequences. This method may develop as a general strategy for stabilizing highly polymorphic peptide regions., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Human prion diseases are classified into sporadic, genetic, and acquired forms. Within this last group, iatrogenic Creutzfeldt-Jakob disease (iCJD) is caused by human-to-human transmission through surgical and medical procedures. After reaching an incidence peak in the 1990s, it is believed that the iCJD historical period is probably coming to an end, thanks to lessons learnt from past infection sources that promoted new prion prevention and decontamination protocols. At this point, we sought to characterise the biomarker profile of iCJD and compare it to that of sporadic CJD (sCJD) for determining the value of available diagnostic tools in promptly recognising iCJD cases. To that end, we collected 23 iCJD samples from seven national CJD surveillance centres and analysed the electroencephalogram and neuroimaging data together with a panel of seven CSF biomarkers: 14-3-3, total tau, phosphorylated/total tau ratio, alpha-synuclein, neurofilament light, YKL-40, and real-time quaking induced conversion of prion protein. Using the cut-off values established for sCJD, we found the sensitivities of these biomarkers for iCJD to be similar to those described for sCJD. Given the limited relevant information on this issue to date, the present study validates the use of current sCJD biomarkers for the diagnosis of future iCJD cases., Competing Interests: The authors declare no conflict of interest.

The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta-2 microglobulin (β2m), which, when accumulated in patients, gives rise to dialysis-related amyloidosis. Interestingly, although the physiologic concentration of β2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine β2m (mβ2m) not only displays a lower amyloid propensity both in vivo and in vitro but also inhibits the aggregation of human β2m in vitro. Here, we compared human and mβ2m for their aggregation propensity, ability to form soluble oligomers, stability, three-dimensional structure and dynamics. Our results indicate that mβ2m low-aggregation propensity is due to two concomitant aspects: the low-aggregation propensity of its primary sequence combined with the absence of high-energy amyloid-competent conformations under native conditions. The identification of the specific properties determining the low-aggregation propensity of mouse β2m will help delineate the molecular risk factors which cause a folded protein to aggregate., (© 2019 Federation of European Biochemical Societies.)

Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), atypical parkinsonisms, frontotemporal dementia (FTLD) and prion diseases are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Although the cause for the initiation of protein aggregation is not well understood, these aggregates are disease-specific. For instance, AD is characterized by the intraneuronal accumulation of tau and extracellular deposition of amyloid-β (Aβ), PD is marked by the intraneuronal accumulation of α-synuclein, many FTLD are associated with the accumulation of TDP-43 while prion diseases show aggregates of misfolded prion protein. Hence, misfolded proteins are considered disease-specific biomarkers and their identification and localization in the CNS, collected postmortem, is required for a definitive diagnosis. With the development of two innovative cell-free amplification techniques named Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking-Induced Conversion (RT-QuIC), traces of disease-specific biomarkers were found in CSF and other peripheral tissues (e.g., urine, blood, and olfactory mucosa) of patients with different NDs. These techniques exploit an important feature shared by many misfolded proteins, that is their ability to interact with their normally folded counterparts and force them to undergo similar structural rearrangements. Essentially, RT-QuIC and PMCA mimic in vitro the same pathological processes of protein misfolding which occur in vivo in a very rapid manner. For this reason, they have been employed for studying different aspects of protein misfolding but, overall, they seem to be very promising for the premortem diagnosis of NDs., (© 2020 Elsevier Inc. All rights reserved.)

Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease.

Prion diseases are a group of neurodegenerative disorders associated with the conformational conversion of the cellular prion protein (PrP C ) into an abnormal misfolded form named PrP Sc . Other than accumulating in the brain, PrP Sc can bind PrP C and force it to change conformation to PrP Sc . The exact mechanism which underlies the process of PrP C /PrP Sc conversion still needs to be defined and many molecules or cofactors might be involved. Several studies have documented an important role of PrP C to act as receptor for abnormally folded forms of α-synuclein which are responsible of a group of diseases known as synucleinopathies. The presence of PrP C was required to promote efficient internalization and spreading of abnormal α-synuclein between cells. In this work, we have assessed whether α-synuclein exerts any role in PrP Sc conversion and propagation either in vitro or in vivo. Indeed, understanding the mechanism of PrP C /PrP Sc conversion and the identification of cofactors involved in this process is crucial for developing new therapeutic strategies. Our results showed that PrP Sc was able to efficiently propagate in the brain of animals even in the absence of α-synuclein thus suggesting that this protein did not act as key modulator of prion propagation. Thus, α-synuclein might take part in this process but is not specifically required for sustaining prion conversion and propagation.

Introduction : Human prion diseases are rare fatal neurodegenerative diseases caused by the misfolding and aggregation of the prion protein in the form of infectious prions. So far, these diseases are incurable. One of the major difficulties in identifying suitable drugs is the availability of robust preclinical screening methods. All molecules identified have been screened using cell-based assays and in vivo murine models. The existence of a continuum of prion strains has hampered the identification of efficacious molecules modulating the progression of different forms of the disease. Areas covered : The advent of new in vitro screening methodologies is allowing for novel strategies to develop new compounds that could interfere with a broad range of diseases. In particular, two innovative techniques named Real Time Quaking Induced Conversion (RT-QuIC) and Protein Misfolding Cyclic Amplification (PMCA) have opened new venues for testing compounds in a rapid a reproducible way. These are discussed within. Expert opinion : For human prion diseases, one major hurdle has been a well-defined screening methodology. In other animal species, cell-based assays have been employed that could replicate animal prions indefinitely. Such a tool for human prion diseases is still missing. Therefore, the advent of RT-QuIC and PMCA has proven instrumental to overcome this limitation.

Prion diseases are fatal neurodegenerative disorders characterized by the cellular prion protein (PrP C ) conversion into a misfolded and infectious isoform termed prion or PrP Sc . The neuropathological mechanism underlying prion toxicity is still unclear, and the debate on prion protein gain- or loss-of-function is still open. PrP C participates to a plethora of physiological mechanisms. For instance, PrP C and copper cooperatively modulate N-methyl-D-aspartate receptor (NMDAR) activity by mediating S-nitrosylation, an inhibitory post-translational modification, hence protecting neurons from excitotoxicity. Here, NMDAR S-nitrosylation levels were biochemically investigated at pre- and post-symptomatic stages of mice intracerebrally inoculated with RML, 139A, and ME7 prion strains. Neuropathological aspects of prion disease were studied by histological analysis and proteinase K digestion. We report that hippocampal NMDAR S-nitrosylation is greatly reduced in all three prion strain infections in both pre-symptomatic and terminal stages of mouse disease. Indeed, we show that NMDAR S-nitrosylation dysregulation affecting prion-inoculated animals precedes the appearance of clinical signs of disease and visible neuropathological changes, such as PrP Sc accumulation and deposition. The pre-symptomatic reduction of NMDAR S-nitrosylation in prion-infected mice may be a possible cause of neuronal death in prion pathology, and it might contribute to the pathology progression opening new therapeutic strategies against prion disorders.

Background: Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of α-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM)., Methods: We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant α-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce α-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP., Results: Our results showed that a significant percentage of MSA and PD samples induced α-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination., Conclusions: Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for α-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

Prion pathologies are characterized by the conformational conversion of the cellular prion protein (PrP C ) into a pathological infectious isoform, known as PrP Sc . 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 PrP Sc 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 (PrP Sc -M) and the other characterized by a predominance of the diglycosylated one (PrP Sc -D). Neuropathological, biochemical, and biophysical assays confirmed that these PrP Sc possess distinctive characteristics. Finally, PMCA analysis of PrP Sc -M and PrP Sc -D generated PrP Sc (PrP Sc -PMCA) whose biophysical properties were different from those of both inocula, suggesting that PMCA selectively amplified a third PrP Sc 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.

The presence of abnormal, disease-related prion protein (PrP D ) has recently been demonstrated by protein misfolding cyclic amplification (PMCA) in urine of patients affected with variant Creutzfeldt-Jakob disease (vCJD), a prion disease typically acquired from consumption of prion contaminated bovine meat. The complexity and multistage process of urine excretion along with the obligatory use of PMCA raise the issue of whether strain characteristics of the PrP D present in vCJD brains, such as infectivity and phenotype determination, are maintained in urine excreted PrP D and following amplification by PMCA. We inoculated transgenic mice expressing normal human PrP with amplified urine and brain homogenate achieving the same 100% attack rate, similar incubation periods (in both cases extremely long) and histopathological features as for type and severity of the lesions. Furthermore, PrP D characteristics analyzed by immunoblot and conformational stability immunoassay were indistinguishable. Inoculation of raw vCJD urine caused no disease, confirming the extremely low concentration of PrP D in vCJD urine. These findings show that strain characteristics of vCJD brain PrP D , including infectivity, are preserved in PrP D present in urine and are faithfully amplified by means of PMCA; moreover, they suggest that the PrP D urine test might allow for the diagnosis and identification of disease subtype also in sporadic CJD.

Beta-2 microglobulin (β2m) is a protein responsible for a pathologic condition, known as dialysis-related amyloidosis (DRA), caused by its aggregation and subsequent amyloid formation. A naturally occurring mutation of β2m, D76N, presents a higher amyloidogenic propensity compared to the wild type counterpart. Since the three-dimensional structure of the protein is essentially unaffected by the mutation, the increased aggregation propensity of D76N has been generally ascribed to its lower thermodynamic stability and increased dynamics. In this study we compare the equilibrium unfolding and the aggregation propensity of wild type β2m and D76N variant at different experimental conditions. Our data revealed a surprising effect of the D76N mutation in the residual structure of the denatured state, which appears less compact than that of the wild type protein. A careful investigation of the structural malleability of the denatured state of wild type β2m and D76N pinpoint a clear role of the denatured state in triggering the amyloidogenic propensity of the protein. The experimental results are discussed in the light of the previous work on β2m and its role in disease.

Prion diseases are neurodegenerative disorders which are caused by an accumulation of the abnormal, misfolded prion protein known as scrapie prion protein (PrP Sc ). These disorders are unique as they occur as sporadic, genetic and acquired forms. Sporadic Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease, accounting for approximately 85-90% of cases, whereas autosomal dominant genetic forms, due to mutations in the prion protein gene (PRNP), account for 10-15% of cases. Genetic forms show a striking variability in their clinical and neuropathological picture and can sometimes mimic other neurodegenerative diseases.We report a novel PRNP mutation (V189I) in four CJD patients from three unrelated pedigrees. In three patients, the clinical features were typical for CJD and the diagnosis was pathologically confirmed, while the fourth patient presented with a complex phenotype including rapidly progressive dementia, behavioral abnormalities, ataxia and extrapyramidal features, and the diagnosis was probable CJD by current criteria, on the basis of PrP Sc detection in CSF by Real Time Quaking-Induced Conversion assay. In all the three patients with autopsy findings, the neuropathological analysis revealed diffuse synaptic type deposition of proteinase K-resistant prion protein (PrP res ), and type 1 PrP res was identified in the brain by western blot analysis. So, the histopathological and biochemical profile associated with the V189I mutation was indistinguishable from the MM1/MV1 subtype of sporadic CJD.Our findings support a pathogenic role for the V189I PRNP variant, confirm the heterogeneity of the clinical phenotypes associated to PRNP mutations and highlight the importance of PrP Sc detection assays as diagnostic tools to unveil prion diseases presenting with atypical phenotypes.

Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC 50 values toward GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 μM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs.

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.

Protein misfolding and aggregation is a central feature of several neurodegenerative disorders including Alzheimer's disease (AD), in which assemblies of amyloid β (Aβ) peptides accumulate in the brain in the form of parenchymal and/or vascular amyloid. A widely accepted concept is that AD is characterized by distinct clinical and neuropathological phenotypes. Recent studies revealed that Aβ assemblies might have structural differences among AD brains and that such pleomorphic assemblies can correlate with distinct disease phenotypes. We found that in both sporadic and inherited forms of AD, amyloid aggregates differ in the biochemical composition of Aβ species. These differences affect the physicochemical properties of Aβ assemblies including aggregation kinetics, resistance to degradation by proteases and seeding ability. Aβ-amyloidosis can be induced and propagated in animal models by inoculation of brain extracts containing aggregated Aβ. We found that brain homogenates from AD patients with different molecular profiles of Aβ are able to induce distinct patterns of Aβ-amyloidosis when injected into mice. Overall these data suggest that the assembly of mixtures of Aβ peptides into different Aβ seeds leads to the formation of distinct subtypes of amyloid having distinctive physicochemical and biological properties which result in the generation of distinct AD molecular subgroups.

Background: Hemoglobin is the major protein found in erythrocytes, where it acts as an oxygen carrier molecule. In recent years, its expression has been reported also in neurons and glial cells, although its role in brain tissue remains still unknown. Altered hemoglobin expression has been associated with various neurodegenerative disorders. Here, we investigated hemoglobin mRNA levels in brains of patients affected by variant, iatrogenic, and sporadic forms of Creutzfeldt-Jakob disease (vCJD, iCJD, sCJD, respectively) and in different genetic forms of prion diseases (gPrD) in comparison to Alzheimer's disease (AD) subjects and age-matched controls. Methods: Total RNA was obtained from the frontal cortex of vCJD ( n = 20), iCJD ( n = 11), sCJD ( n = 23), gPrD ( n = 30), and AD ( n = 14) patients and age-matched controls ( n = 30). RT-qPCR was performed for hemoglobin transcripts HBB and HBA1/2 using four reference genes for normalization. In addition, expression analysis of the specific erythrocyte marker ALAS2 was performed in order to account for blood contamination of the tissue samples. Hba1/2 and Hbb protein expression was then investigated with immunofluorescence and confocal microscope analysis. Results: We observed a significant up-regulation of HBA1/2 in vCJD brains together with a significant down-regulation of HBB in iCJD. In addition, while in sporadic and genetic forms of prion disease hemoglobin transcripts did not shown any alterations, both chains display a strong down-regulation in AD brains. These results were confirmed also at a protein level. Conclusions: These data indicate distinct hemoglobin transcriptional responses depending on the specific alterations occurring in different neurodegenerative diseases. In particular, the initial site of misfolding event (central nervous system vs. peripheral tissue)-together with specific molecular and conformational features of the pathological agent of the disease-seem to dictate the peculiar hemoglobin dysregulation found in prion and non-prion neurodegenerative disorders. In addition, these results suggest that gene expression of HBB and HBA1/2 in brain tissue is differentially affected by distinct prion and prion-like aggregating protein strains. Validation of these results in more accessible tissues could prompt the development of novel diagnostic tests for neurodegenerative disorders.

An early and accurate in vivo diagnosis of rapidly progressive dementia remains challenging, despite its critical importance for the outcome of treatable forms, and the formulation of prognosis. Real-Time Quaking-Induced Conversion (RT-QuIC) is an in vitro assay that, for the first time, specifically discriminates patients with prion disease. Here, using cerebrospinal fluid (CSF) samples from 239 patients with definite or probable prion disease and 100 patients with a definite alternative diagnosis, we compared the performance of the first (PQ-CSF) and second generation (IQ-CSF) RT-QuIC assays, and investigated the diagnostic value of IQ-CSF across the broad spectrum of human prions. Our results confirm the high sensitivity of IQ-CSF for detecting human prions with a sub-optimal sensitivity for the sporadic CJD subtypes MM2C and MM2T, and a low sensitivity limited to variant CJD, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. While we found no difference in specificity between PQ-CSF and IQ-CSF, the latter showed a significant improvement in sensitivity, allowing prion detection in about 80% of PQ-CSF negative CJD samples. Our results strongly support the implementation of IQ-CSF in clinical practice. By rapidly confirming or excluding CJD with high accuracy the assay is expected to improve the outcome for patients and their enrollment in therapeutic trials.

The precise molecular mechanism of how misfolded α-synuclein (α-Syn) accumulates and spreads in synucleinopathies is still unknown. Here, we show the role of the cellular prion protein (PrP C ) in mediating the uptake and the spread of recombinant α-Syn amyloids. The in vitro data revealed that the presence of PrP C fosters the higher uptake of α-Syn amyloid fibrils, which was also confirmed in vivo in wild type (Prnp +/+ ) compared to PrP knock-out (Prnp -/- ) mice. Additionally, the presence of α-Syn amyloids blocked the replication of scrapie prions (PrP Sc ) in vitro and ex vivo, indicating a link between the two proteins. Indeed, whilst PrP C is mediating the internalization of α-Syn amyloids, PrP Sc is not able to replicate in their presence. This observation has pathological relevance, since several reported case studies show that the accumulation of α-Syn amyloid deposits in Creutzfeldt-Jakob disease patients is accompanied by a longer disease course.

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 × 10 -14  g/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%.

Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches. Our results show that a reduction in the expression of this molecular chaperone accelerates prion pathogenesis in vivo. In addition, we observed that prion replication in cell culture was inversely related to the levels of expression of GRP78 and that both proteins interact in the cellular context. Finally, incubation of PrP Sc with recombinant GRP78 led to the dose-dependent reduction of protease-resistant PrP Sc in vitro. Our results uncover a novel role of GRP78 in reducing prion pathogenesis, suggesting that modulating its levels/activity may offer a novel opportunity for designing therapeutic approaches for these diseases. These findings may also have implications for other diseases involving the accumulation of misfolded proteins.

Transmissible spongiform encephalopathies or prion diseases are a group of fatal neurodegenerative diseases caused by unconventional infectious agents, known as prions (PrP Sc ). Prions derive from a conformational conversion of the normally folded prion protein (PrP C ), which acquires pathological and infectious features. Moreover, PrP Sc is able to transmit the pathological conformation to PrP C through a mechanism that is still not well understood. The generation of synthetic prions, which behave like natural prions, is of fundamental importance to study the process of PrP C conversion and to assess the efficacy of therapeutic strategies to interfere with this process. Moreover, the ability of synthetic prions to induce pathology in animals confirms that the pathological properties of the prion strains are all enciphered in abnormal conformations, characterizing these infectious agents., (© 2017 Elsevier Inc. All rights reserved.)

Prions are the infectious agents that cause devastating and untreatable disorders known as Transmissible Spongiform Encephalopathies (TSEs). The pathologic events and the infectious nature of these transmissible agents are not completely understood yet. Due to the difficulties in inactivating prions, working with them requires specific recommendations and precautions. Moreover, with the advent of innovative technologies, such as the Protein Misfolding Cyclic Amplification (PMCA) and the Real Time Quaking-Induced Conversion (RT-QuIC), prions could be amplified in vitro and the infectious features of the amplified products need to be carefully assessed., (© 2017 Elsevier Inc. All rights reserved.)

Transmissible spongiform encephalopathies, or prion diseases, are a group of incurable disorders caused by the accumulation of an abnormally folded prion protein (PrP Sc ) in the brain. According to the "protein-only" hypothesis, PrP Sc is the infectious agent able to propagate the disease by acting as a template for the conversion of the correctly folded prion protein (PrP C ) into the pathological isoform. Recently, the mechanism of PrP C conversion has been mimicked in vitro using an innovative technique named protein misfolding cyclic amplification (PMCA). This technology represents a great tool for studying diverse aspects of prion biology in the field of basic research and diagnosis. Moreover, PMCA can be expanded for the study of the misfolding process associated to other neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and frontotemporal lobar degeneration., (© 2017 Elsevier Inc. All rights reserved.)

Human prion diseases are infectious and invariably fatal neurodegenerative diseases. They include sporadic Creutzfeldt-Jakob disease (sCJD), the most common form, and variant CJD (vCJD), which is caused by interspecies transmission of prions from cattle infected by bovine spongiform encephalopathy. Development of a biochemical assay for the sensitive, specific, early, and noninvasive detection of prions (PrP Sc ) in the blood of patients affected by prion disease is a top medical priority to increase the safety of the blood supply. vCJD has already been transmitted from human to human by blood transfusion, and the number of asymptomatic carriers of vCJD in the U.K. alone is estimated to be 1 in 2000 people. We used the protein misfolding cyclic amplification (PMCA) technique to analyze blood samples from 14 cases of vCJD and 153 controls, including patients affected by sCJD and other neurodegenerative or neurological disorders as well as healthy subjects. Our results showed that PrP Sc could be detected with 100% sensitivity and specificity in blood samples from vCJD patients. Detection was possible in any of the blood fractions analyzed and could be done with as little as a few microliters of sample volume. The PrP Sc concentration in blood was estimated to be ~0.5 pg/ml. Our findings suggest that PMCA may be useful for premortem noninvasive diagnosis of vCJD and to identify prion contamination of the blood supply. Further studies are needed to fully validate the technology., (Copyright © 2016, American Association for the Advancement of Science.)