Diagnostic Accuracy of Prion Disease Biomarkers in Iatrogenic Creutzfeldt-Jakob Disease

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.This research was funded by the Instituto Carlos III (grants CP/00041 and PI19/00144) and by the Fundació La Marató de TV3 (201821‐30‐31‐32) to FL and by the Robert Koch Institute through funds from the Federal Ministry of Health (grant No, 1369‐341) to IZ. This project was also funded at 65% by the Fondo Europeo de Desarrollo Regional (FEDER) through the Interreg V‐A España‐Francia‐Andorra (POCTEFA 2014‐2020) programme. SJC is funded in part by a NHMRC Practitioner Fellowship (identification #APP1105784).S

Cerebrospinal fluid total prion protein in the spectrum of prion diseases

Cerebrospinal fluid (CSF) total prion protein (t-PrP) is decreased in sporadic Creutzfeldt-Jakob disease (sCJD). However, data on the comparative signatures of t-PrP across the spectrum of prion diseases, longitudinal changes during disease progression, and levels in pre-clinical cases are scarce. T-PrP was quantified in neurological diseases (ND, n = 147) and in prion diseases from different aetiologies including sporadic (sCJD, n = 193), iatrogenic (iCJD, n = 12) and genetic (n = 209) forms. T-PrP was also measured in serial lumbar punctures obtained from sCJD cases at different symptomatic disease stages, and in asymptomatic prion protein gene (PRNP) mutation carriers. Compared to ND, t-PrP concentrations were significantly decreased in sCJD, iCJD and in genetic prion diseases associated with the three most common mutations E200K, V210I (associated with genetic CJD) and D178N-129M (associated with fatal familial insomnia). In contrast, t-PrP concentrations in P102L mutants (associated with the Gerstmann-Sträussler-Scheinker syndrome) remained unaltered. In serial lumbar punctures obtained at different disease stages of sCJD patients, t-PrP concentrations inversely correlated with disease progression. Decreased mean t-PrP values were detected in asymptomatic D178-129M mutant carriers, but not in E200K and P102L carriers. The presence of low CSF t-PrP is common to all types of prion diseases regardless of their aetiology albeit with mutation-specific exceptions in a minority of genetic cases. In some genetic prion disease, decreased levels are already detected at pre-clinical stages and diminish in parallel with disease progression. Our data indicate that CSF t-PrP concentrations may have a role as a pre-clinical or early symptomatic diagnostic biomarker in prion diseases as well as in the evaluation of therapeutic interventions

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt–jakob disease:An international study

Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt\u2013Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160\u2013165

Identification of novel risk loci and causal insights for sporadic Creutzfeldt-Jakob disease: a genome-wide association study

Background: Human prion diseases are rare and usually rapidly fatal neurodegenerative disorders, the most common being sporadic Creutzfeldt-Jakob disease (sCJD). Variants in the PRNP gene that encodes prion protein are strong risk factors for sCJD but, although the condition has similar heritability to other neurodegenerative disorders, no other genetic risk loci have been confirmed. We aimed to discover new genetic risk factors for sCJD, and their causal mechanisms. Methods: We did a genome-wide association study of sCJD in European ancestry populations (patients diagnosed with probable or definite sCJD identified at national CJD referral centres) with a two-stage study design using genotyping arrays and exome sequencing. Conditional, transcriptional, and histological analyses of implicated genes and proteins in brain tissues, and tests of the effects of risk variants on clinical phenotypes, were done using deep longitudinal clinical cohort data. Control data from healthy individuals were obtained from publicly available datasets matched for country. Findings: Samples from 5208 cases were obtained between 1990 and 2014. We found 41 genome-wide significant single nucleotide polymorphisms (SNPs) and independently replicated findings at three loci associated with sCJD risk; within PRNP (rs1799990; additive model odds ratio [OR] 1·23 [95% CI 1·17-1·30], p=2·68 × 10-15; heterozygous model p=1·01 × 10-135), STX6 (rs3747957; OR 1·16 [1·10-1·22], p=9·74 × 10-9), and GAL3ST1 (rs2267161; OR 1·18 [1·12-1·25], p=8·60 × 10-10). Follow-up analyses showed that associations at PRNP and GAL3ST1 are likely to be caused by common variants that alter the protein sequence, whereas risk variants in STX6 are associated with increased expression of the major transcripts in disease-relevant brain regions. Interpretation: We present, to our knowledge, the first evidence of statistically robust genetic associations in sporadic human prion disease that implicate intracellular trafficking and sphingolipid metabolism as molecular causal mechanisms. Risk SNPs in STX6 are shared with progressive supranuclear palsy, a neurodegenerative disease associated with misfolding of protein tau, indicating that sCJD might share the same causal mechanisms as prion-like disorders. Funding: Medical Research Council and the UK National Institute of Health Research in part through the Biomedical Research Centre at University College London Hospitals National Health Service Foundation Trust

Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species.

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies

Crude M1000 brain homogenate acute synaptotoxicity dose-response relationships and estimation of total PrP and PK-resistant PrP^Sc in preparations used in electrophysiology experiments.

<p>A dilution series of recombinant full-length mouse PrP was western blotted (probed with 8H4 monoclonal antibody) and analysed with densitometry to generate a standard curve with aliquots of preparations utilised in electrophysiology experiments run on the same gel. (A) Quantification of levels of total PrP species (without PK treatment) in 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000, as well as in 0.5% dM1000 (PrP immuno-depleted cM1000). (B) Relative levels of at least modestly PK-resistant PrP^Sc (after treatment with 5μg/mL PK for 1 hour at 37°C) in 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000, as well as in 0.5% (w/v) PK+M1000 and 0.5% (w/v) PK+IP-M1000. (C & D) Percentage of LTP and PTP impairments (calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007214#sec002" target="_blank">Methods</a>) following exposure of 12-week old WT hippocampal slices to non-PK treated 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000 compared to 0.5% (w/v) cNBH controls (One-way ANOVA with Bonferroni correction for multiple comparisons [LTP: <i>p<0</i>.<i>0001</i>; PTP: <i>p<0</i>.<i>0001</i>]). In 0.5% (w/v) cM1000, LTP and PTP were significantly impaired by 53 ± 9% and 30 ± 6%, respectively. In 0.25% (w/v) cM1000, LTP and PTP were significantly impaired by 30 ± 2% and 26 ± 7%, respectively. In 0.1% (w/v) cM1000, LTP and PTP were not significantly impaired, reduced by only 19 ± 6% and 0.1 ± 9%, respectively. Percentages of LTP and PTP impairment in WT hippocampal slices following exposure to 0.5% dM1000, PK+M1000, and PK+IP-M1000 were also compared to 0.5% (w/v) cNBH controls (One-way ANOVA with Bonferroni correction for multiple comparisons [LTP: <i>p<0</i>.<i>0001</i>; PTP: <i>p<0</i>.<i>0039]</i>). In 0.5% (w/v) dM1000, the LTP was not impaired significantly (reduced by 14 ± 7%) while PTP was significantly impaired by 38 ± 3%. In 0.5% (w/v) PK+M1000, LTP and PTP were significantly impaired by 48 ± 7% and 26 ± 7%, respectively. In 0.5% (w/v) PK+IP-M1000, LTP and PTP were significantly impaired by 59 ± 5% and 36 ± 5%, respectively. (E & F) Percentage LTP and PTP impairments following exposure of 12-week old PrPo/o hippocampal slices to non-PK treated 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000 compared to 0.5% (w/v) cNBH controls (One-way ANOVA with Bonferroni correction for multiple comparisons [LTP: <i>p<0</i>.<i>0002</i>; PTP: <i>p<0</i>.<i>0027]</i>). In 0.5% (w/v) cM1000, LTP and PTP were significantly impaired by 58 ± 8% and 32 ± 8%, respectively. In 0.25% (w/v) cM1000, LTP and PTP were significantly impaired by 48 ± 10% and 39 ± 6%, respectively. In 0.1% (w/v) cM1000, LTP and PTP were not significantly impaired (reduced by only 15 ± 7% and 13 ± 5%, respectively). Data are presented as ± SEM. *<i>p<0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i>, ***<i>p<0</i>.<i>001</i>, ****<i>p<0</i>.<i>0001</i>.</p

Crude ex vivo PrP^Sc containing preparations acutely impair LTP.

<p>(A) Western blot of cNBH not treated (-) or treated (+) with 50μg/ml PK. The PK- cNBH was utilised as control preparations for the treatments of WT hippocampal slices using prion-containing crude brain homogenates. (B) cM1000 superfused for 5 minutes over hippocampal slices from 12-week old WT mice approximately 20 minutes prior to HFS caused a significant impairment of LTP with (C) average LTP reduced by 53 ± 9% (n = 6) compared to cNBH. (<i>p = 0</i>.<i>0087</i>). (D) Western blot of cM1000 not treated (-) or treated (+) with 50μg/ml PK with PK+ proving presence of PrP^Sc. (E) cMU02 superfused for 5 minutes over slices from 12-week old WT mice approximately 20 minutes prior to HFS caused a significant impairment of LTP with (F) average LTP reduced by 62 ± 19% (n = 6) compared to cNBH. (<i>p = 0</i>.<i>0129</i>). (G) Western blot of cMU02 not treated (-) or treated (+) with 50μg/ml with PK+ proving presence of PrP^Sc. (H) The PK- cM1000 was superfused for 5 minutes over slices from 11-month old WT mice approximately 20 minutes prior to HFS caused significant impairment of LTP with (I) average LTP reduced by 44 ± 7%; (n = 7) compared to cNBH. (<i>p = 0</i>.<i>0006</i>). (J) Western blot of MoRK13-Inf not treated (-) or treated (+) with 50μg/ml PK with PK+ proving presence of PrP^Sc. (K) The PK- MoRK13-Inf was superfused for 5 minutes over slices from 11-month old WT mice approximately 20 minutes prior to HFS caused significant impairment of LTP with (L) average LTP reduced by 40 ± 6% (n = 6) compared to MoRK13-Un (<i>p = 0</i>.<i>0172</i>). (B, E, H & K) The first five-minute fEPSP recordings following HFS trains have been removed to enhance clarity and the last 10 minutes of post-HFS recordings were used for LTP analysis. (A, D, G, J) Molecular markers are provided at left. (B, E, H, K) Examples of raw fEPSP traces are provided as insets. Data are presented as ± SEM. *<i>p<0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i>, ***<i>p<0</i>.<i>001</i>, ****<i>p<0</i>.<i>0001</i>.</p

Altered expression levels of key pre- and post-synaptic markers correlate with synaptic dysfunction in WT hippocampi.

<p>Following induction of LTP, hippocampi were homogenized and biochemically analysed for key synaptic markers. (A & D) Hippocampi treated with cM1000 [<i>c</i>] expressed reduced levels of pre-synaptic markers, synaptophysin (<i>p = 0</i>.<i>0280</i>) and VGLUT1 (<i>p = 0</i>.<i>0108</i>), relative to those treated with cNBH [<i>c</i>]. Conversely, WT hippocampi treated with dM1000 [<i>d</i>] were associated with unaltered synaptophysin and VGLUT1 levels relative dNBH [<i>d</i>]. (B & D) NR2A/B-containing NMDAR were also reduced in hippocampi treated with cM1000 (NR2A: <i>p = 0</i>.<i>0012</i>; NR2B: <i>p = 0</i>.<i>0003</i>) relative to those treated with cNBH, with unaltered levels when employing dM1000 and dNBH. GluA2-containing AMPAR were also reduced in hippocampi treated with cM1000 relative to those treated with cNBH (<i>p = 0</i>.<i>0077)</i>, with unaltered levels when employing dM1000 and dNBH. (C & D) Expression levels of pERK and pCREB were reduced in slices exposed to cM1000 (pERK: <i>p = 0</i>.<i>0339</i>; pCREB: <i>p = 0</i>.<i>0108</i>) relative to cNBH but were unaltered when employing dM1000 and dNBH. PSD95 remained unaltered after exposure to both cM1000 and dM1000 supporting no loss of pre-existing synapses in prion acute synaptotoxicity. Fyn levels remained unaffected after treatment with either cM1000 or dM1000. (E & F) Relative to cNBH, cM1000 reduced expression levels of procaspase 3 (<i>p = 0</i>.<i>0028</i>) with unaltered production of the active form, thereby significantly reducing the pro- to active caspase 3 ratio (<i>p = 0</i>.<i>0083</i>). The pro-caspase 3 expression level was unaltered after treatment with dM1000 compared with dNBH with a normal pro- to active caspase 3 ratio. (A-C, E) Molecular weight markers are provided at right. Data are presented as ± SEM. *<i>p<0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i>, ***<i>p<0</i>.<i>001</i>, ****<i>p<0</i>.<i>0001</i>.</p

Prion acute synaptotoxicity demonstrates enhanced pre-synaptic vulnerability in WT hippocampal slices.

<p>(A) Normal PPF ratio reductions (the ratio becomes significantly reduced in PPF2 relative to PPF1) were obtained in slices after LTP induction and exposure to all negative controls: cNBH (for cM1000 treatments in 12-week old [<i>p<0001</i>] and 11-month old mice [<i>p = 0</i>.<i>0008</i>]; cMU02 treatment [<i>p = 0</i>.<i>0104</i>]); dNBH (<i>p = 0</i>.<i>0017)</i>; PK+NBH (<i>p = 0</i>.<i>0182</i>); PK+IP-NBH (<i>p = 0</i>.<i>0002</i>); and MoRK13-Un (<i>p = 0</i>.<i>0170</i>). Conversely, PPF ratio in slices treated with cM1000 (both in 12-week and 11-month old mice), cMU02, MoRK13-Inf, PK+M1000, and PK+IP-M1000 was not reduced in PPF2 relative to PPF1, showing poor <i>Pr</i> associated with the LTP disruption. The PPF ratio was normal in dM1000 (<i>p = 0</i>.<i>0013</i>) concomitant with the recovery of LTP. (B) Treatment of slices with cM1000 without inducing LTP expression did not affect PPF ratios relative to slices treated with cNBH. (C) Relative to appropriate negative controls, PTP was significantly disrupted after cM1000 treatment of slices from 12-week-old (<i>p = 0</i>.<i>0144</i>) and 11-month-old (<i>p = 0</i>.<i>0006</i>) mice. Similar PTP disruption was obtained following treatment with cMU02 (<i>p = 0</i>.<i>0391</i>), MoRK13-Inf (<i>p = 0</i>.<i>0172</i>), dM1000 (<i>p</i> = 0.0062), PK+M1000 (<i>p = 0</i>.<i>0359</i>), and PK+IP-M1000 (<i>p<0</i>.<i>0001</i>). (D) The <i>Pr</i> during the HFS trains was normal in both cM1000 and dM1000 relative to cNBH and dNBH controls, respectively where the <i>Pr</i> became significantly increased (determined by the P1 to P2 ratio in each train) in T2 and T3 relative to T1. (E & F) Consistent with the normal <i>Pr</i>, the rate of RRP depletion was normal across three HFS trains in both cM1000 (E) and dM1000 (F), where the time constant of decay between P3 and P9 in each train was not different between cNBH and cM1000, as well as between dNBH and dM1000. (G & H) The size of RRP (determined by the Y-intercepts of the linear fit of the last four pulses of HFS) became significantly diminished at T3 in cM1000 (G) and dM1000 (H) relative to cNBH (G) and dNBH (H). (I & J) This reduction in RRP size was caused by a significant impairment of the RRP replenishment (indicated by the increase in the RRP size between trains) to refill the RRP at T3 in both cM1000 (I) and dM1000 (J) compared with cNBH (I) and dNBH (J). The reduction in RRP size contributed directly to the impairment of PTP. (A-D) Examples of raw fEPSP traces are provided as insets. Data are presented as ± SEM. *<i>p<0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i>, ***<i>p<0</i>.<i>001</i>, ****<i>p<0</i>.<i>0001</i>.</p

PK-resistant PrP species are responsible for acute synaptotoxicity.

<p>(A) PrP immuno-depletion selectively depleted ~77 ± 12% of PrP^C (dNBH) from cNBH and ~77 ± 9% total PrP species and 96 ± 4% of PK-resistant PrP (dM1000) from cM1000 relative to the normal rabbit serum (NRS) controls. Controls and depleted preparations were either not treated (-) or treated (+) with 50μg/ml PK to determine the level of PK-resistant PrP. (B) dM1000 superfused for 5 minutes over hippocampal slices from 12-week old WT mice approximately 20 minutes prior to HFS trains displayed significant rescue of LTP with (C) average LTP increased 74 ± 14% (n = 8), which was no longer significantly different to slices superfused with dNBH. (D) Western blot of cNBH and cM1000 treated with 5μg/mL PK (PK+NBH; PK+M1000), which was sufficient to completely degrade all PrP^C in cNBH. (E) PK+M1000 superfused for 5 minutes over slices from 12-week old WT mice approximately 20 minutes prior to HFS significantly impaired LTP with (F) average LTP reduced by 48 ± 7% (n = 8) relative to PK+NBH preparations (<i>p = 0</i>.<i>003</i>). (G) Pellets generated through PrP immuno-precipitation (IP) of total PrP from cNBH and cM1000 were resuspended and western blotted after 5μg/mL PK digestion to specifically elute at least modestly PK resistant PrP^Sc, with comparison made to NBH and M1000 resuspended pellets wherein PK-resistant PrP was eluted by boiling after PK digestion with 5 μg/ml PK. (H) The resuspended, PK-eluted IP pellets from cNBH (PK+IP-NBH) or cM1000 (PK+IP-M1000) superfused for 5 minutes over slices from 12-week old WT mice approximately 20 minutes prior to HFS caused significant impairment of LTP measured during the last 10-minutes of post-HFS recording, with (I) average LTP reduced by 59 ± 5% (n = 8) relative to PK+IP-NBH preparations (<i>p = 0</i>.<i>001</i>). (B, E & H) The first five-minute fEPSP recordings following HFS trains have been removed to enhance clarity. (A: lower panel, D, G) Molecular markers are provided at left. (B, E, H) Examples of raw fEPSP traces are provided as insets. Data are presented as ± SEM. *<i>p<0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i>, ***<i>p<0</i>.<i>001</i>, ****<i>p<0</i>.<i>0001</i>.</p