Saima Zafar

Characteristic CSF prion seeding efficiency in humans with prion diseases.

The development of in vitro amplification systems allows detecting femtomolar amounts of prion protein scrapie (PrPSc) in human cerebrospinal fluid (CSF). We performed a CSF study to determine the effects of prion disease type, codon 129 genotype, PrPSc type, and other disease-related factors on the real-time quaking-induced conversion (RT-QuIC) response. We analyzed times to 10,000 relative fluorescence units, areas under the curve and the signal maximum of RT-QuIC response as seeding parameters of interest. Interestingly, type of prion disease (sporadic vs. genetic) and the PRNP mutation (E200K vs. V210I and FFI), codon 129 genotype, and PrPSc type affected RT-QuIC response. In genetic forms, type of mutation showed the strongest effect on the observed outcome variables. In sporadic CJD, MM1 patients displayed a higher RT-QuIC signal maximum compared to MV1 and VV1. Age and gender were not associated with RT-QuIC signal, but patients with a short disease course showed a higher seeding efficiency of the RT-QuIC response. This study demonstrated that PrPSc characteristics in the CSF of human prion disease patients are associated with disease subtypes and rate of decline as defined by disease duration.

Proteomics Approach to Identify the Interacting Partners of Cellular Prion Protein and Characterization of Rab7a Interaction in Neuronal Cells

The present study was undertaken to identify proteins interacting with PrP(C) that could provide new insights into its physiological functions and pathological role. Human PrP(C) was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrP(C) along with its interacting proteins were affinity purified using STrEP-Tactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrP(C) in this neuronal cell line. Of these, 15 were already known for their interaction with PrP(C) or PrP(Sc), while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrP(C). siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrP(C) expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrP(C) in Rab9 positive endosomal compartments and consequently an increased colocalization between PrP(C)/Rab9. However, the Rab9 accumulated PrP(C) remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrP(C) and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrP(C).

Detection of Infectivity in Blood of Persons with Variant and Sporadic Creutzfeldt-Jakob Disease

We report the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with variant Creutzfeldt-Jakob disease and in the plasma of 2 in 4 persons whose tests were positive for sporadic Creutzfeldt-Jakob disease. The measured infectivity levels were comparable to those reported in various animals with transmissible spongiform encephalopathies.

Subtype and Regional-Specific Neuroinflammation in Sporadic Creutzfeldt–Jakob Disease

The present study identifies deregulated cytokines and mediators of the immune response in the frontal cortex and cerebellum of sporadic Creutzfeldt–Jakob disease (sCJD) MM1 and VV2 subtypes compared to age-matched controls. Deregulated genes include pro- and anti-inflammatory cytokines, toll-like receptors, colony stimulating factors, cathepsins, members of the complement system, and members of the integrin and CTL/CTLD family with particular regional and sCJD subtype patterns. Analysis of cytokines and mediators at protein level shows expression of selected molecules and receptors in neurons, in astrocytes, and/or in microglia, thus suggesting interactions between neurons and glial cells, mainly microglia, in the neuroinflammatory response in sCJD. Similar inflammatory responses have been shown in the tg340 sCJD MM1 mice, revealing a progressive deregulation of inflammatory mediators with disease progression. Yet, inflammatory molecules involved are subjected to species differences in humans and mice. Moreover, inflammatory-related cell signaling pathways NFκB/IKK and JAK/STAT are activated in sCJD and sCJD MM1 mice. Together, the present observations show a self-sustained complex inflammatory and inflammatory-related responses occurring already at early clinical stages in animal model and dramatically progressing at advanced stages of sCJD. Considering this scenario, measures tailored to modulate (activate or inhibit) specific molecules could be therapeutic options in CJD.

The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases

The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPSc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays—such as protein misfolding cyclic amplification (PMCA)—which are based on sonication, the RT-QuIC technique is based on prion seed–induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimers and Parkinsons disease.

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrPc). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrPc, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

Subtype and regional regulation of prion biomarkers in sporadic Creutzfeldt-Jakob disease.

Creutzfeldt–Jakob disease (CJD) is a rapid progressive neurological disease leading to dementia and death. Prion biomarkers are altered in the cerebrospinal fluid (CSF) of CJD patients, but the pathogenic mechanisms underlying these alterations are still unknown. The present study examined prion biomarker levels in the brain and CSF of sporadic CJD (sCJD) cases and their correlation with neuropathological lesion profiles.

Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases

The present study investigates whether posttranslational modifications of cellular prion protein (PrP(C)) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrP(Sc)) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrP(C) charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrP(C) were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrP(C) isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrP(Sc) type 2 (vs. PrP(Sc) type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrP(C) species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrP(C) in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors.

Quantification of CSF biomarkers using an electrochemiluminescence-based detection system in the differential diagnosis of AD and sCJD

The identification of reliable diagnostic tools for the differential diagnosis between sporadic Creutzfeldt–Jakob Disease (sCJD) and Alzheimer’s disease (AD) remains impeded by the existing clinical, neuropathological and molecular overlap between both diseases. The development of new tools for the quantitative measurement of biomarkers is gaining experimental momentum due to recent advances in high-throughput screening analysis and with the optimization of assays for their quantification in biological fluids, including cerebrospinal fluid (CSF). Electrochemiluminescence (ECL)-based immunoassays have demonstrated to achieve clinical quality performance in a variety of sample types due to its high sensitivity and dynamic range. Here, we quantified the CSF levels of Tau-protein, β-amyloid 1-42 (Aβ42) and α-synuclein, as important biomarkers in CSF used in the differential diagnosis of neurodegenerative disorders in 12 AD, 12 sCJD and 12 control cases by singleplex ECL-based technology. Its performance has been compared to classical enzyme-linked immunosorbent assays (ELISA) to confront their clinical accuracy. ECL-based technology validates previous data obtained with ELISA and presents a higher performance in the discrimination of three analysed groups as determined by increased area under the curve (AUC) values for the three biomarkers. Importantly, α-synuclein levels detected by ECL allow an excellent discrimination between sCJD cases and AD and control cases, unveiling a new clinical approach for the differential diagnosis of sCJD.

Evaluation of α-synuclein as a novel cerebrospinal fluid biomarker in different forms of prion diseases

Accurate diagnosis of prion diseases and discrimination from alternative dementias gain importance in the clinical routine, but partial overlap in cerebrospinal fluid (CSF) biomarkers impedes absolute discrimination in the differential diagnostic context.