PMCA Applications for Prion Detection in Peripheral Tissues of Patients with Variant Creutzfeldt-Jakob Disease

settings Order Article Reprints Open AccessReview PMCA Applications for Prion Detection in Peripheral Tissues of Patients with Variant Creutzfeldt-Jakob Disease by Giorgio Giaccone and Fabio Moda * [ORCID] Fondazione IRCCS Istituto Neurologico Carlo Besta, Division of Neurology 5—Neuropathology, 20133 Milan, Italy * Author to whom correspondence should be addressed. Biomolecules 2020, 10(3), 405; https://doi.org/10.3390/biom10030405 Submission received: 13 February 2020 / Revised: 2 March 2020 / Accepted: 5 March 2020 / Published: 5 March 2020 (This article belongs to the Special Issue Prion Disease Biomarkers: Recent Advances) Download keyboard_arrow_down Versions Notes Abstract 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 PrPSc (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 PrPSc. Thanks to the advent of an innovative technique named protein misfolding cyclic amplification (PMCA), traces of PrPSc, 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

Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-β

We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics

Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease

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 (PrPSc) 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 PrPSc 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 PrPSc 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

Treatment with a non-toxic, self-replicating anti-prion delays or prevents prion disease in vivo

Transmissible spongiform encephalopathies (TSEs) are fatal neurological disorders caused by prions, which are composed of a misfolded protein (PrP Sc) that self-propagates in the brain of infected individuals by converting the normal prion protein (PrP C) into the pathological isoform. Here, we report a novel experimental strategy for preventing prion disease based on producing a self-replicating, but innocuous PrP Sc -like form, termed anti-prion, which can compete with the replication of pathogenic prions. Our results show that a prophylactic inoculation of prion-infected animals with an anti-prion delays the onset of the disease and in some animals completely prevents the development of clinical symptoms and brain damage. The data indicate that a single injection of the anti-prion eliminated ∼99% of the infectivity associated to pathogenic prions. Furthermore, this treatment caused significant changes in the profile of regional PrP Sc deposition in the brains of animals that were treated, but still succumbed to the disease. Our findings provide new insights for a mechanistic understanding of prion replication and support the concept that prion replication can be separated from toxicity, providing a novel target for therapeutic intervention

Secondary Protein Aggregates in Neurodegenerative Diseases: Almost the Rule Rather than the Exception

The presence of protein aggregates is a hallmark of many neurodegenerative diseases, including Parkinson’s disease (PD), Alzheimer’s disease (AD), and frontotemporal lobar degeneration (FTLD). Traditionally, each disease has been associated with the aggregation of specific proteins, which serve as disease-specific biomarkers. For example, aggregates of α-synuclein (α-syn) are found in αsynucleinopathies such as PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Similarly, AD is characterized by aggregates of amyloid-beta (Aβ) and tau proteins. However, it has been observed that these protein aggregates can also occur in other neurodegenerative diseases, contributing to disease progression. For instance, α-syn aggregates have been detected in AD, Down syndrome, Huntington’s disease, prion diseases, and various forms of FTLD. Similarly, Aβ aggregates have been found in conditions like DLB and PD. Tau aggregates, in addition to being present in primary tauopathies, have been identified in prion diseases, α-synucleinopathies, and cognitively healthy aged subjects. Finally, aggregates of TDP-43, typically associated with FTLD and amyotrophic lateral sclerosis (ALS), have been observed in AD, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), MSA, DLB, and other neurodegenerative diseases. These findings highlight the complexity of protein aggregation in neurodegeneration and suggest potential interactions and common mechanisms underlying different diseases. A deeper understating of this complex scenario may eventually lead to the identification of a better elucidation of the pathogenetic mechanisms of these devastating conditions and hopefully new therapeutic stragegies

Sporadic Creutzfeldt-Jakob disease: Real-Time Quaking Induced Conversion (RT-QuIC) assay represents a major diagnostic advance

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

Sporadic MM-1 Type Creutzfeldt-Jakob disease with hemiballic presentation and no cognitive impairment until death: How New NCJDRSU diagnostic criteria may allow early diagnosis

Sporadic Creutzfeldt-Jakob disease is the most common human prion disorder. Although associated with heterogeneous clinical phenotypes, its distinctive feature is the presence of a rapidly progressive multidomain cognitive impairment. We describe the atypical case of a patient affected by sporadic Methionine/Methionine type 1 Creutzfeldt-Jakob disease (typically associated with early cognitive decline) who presented with an isolated hemiballic syndrome and no signs of cognitive involvement until death. We review sporadic Creutzfeldt-Jakob disease diagnostic criteria and their updates since their first formulation, highlighting their limitations in clinical diagnostic work-up. Finally, we discuss the recently introduced National Creutzfeldt-Jakob Disease Research and Surveillance Unit diagnostic criteria, suggesting how their application could support an early clinical diagnosis, even in atypical cases, such as the one presented

Defined \u3b1-synuclein prion-like molecular assemblies spreading in cell culture

BACKGROUND: \u3b1-Synuclein (\u3b1-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse experiments suggest intercellular \u3b1-syn transfer. RESULTS: Through a methodology used to obtain synthetic mammalian prions, we tested whether recombinant human \u3b1-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human \u3b1-syn was sufficient to induce aggregation of endogenous \u3b1-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type \u3b1-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required. CONCLUSIONS: Our results provide compelling evidence that endogenous \u3b1-syn can accumulate in cell culture after a single exposure to exogenous \u3b1-syn short amyloid fibrils. Importantly, using \u3b1-syn short amyloid fibrils as seed, endogenous \u3b1-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic \u3b1-syn aggregates

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrPC) into disease-specific forms, termed PrP Sc, that have the ability to interact with PrPC promoting its conversion to PrPSc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrPC region involved in the interaction with PrPSc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrPSc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding