α-Synuclein seed amplification assay detects Lewy body co-pathology in autosomal dominant Alzheimer's disease late in the disease course and dependent on Lewy pathology burden.

INTRODUCTION: Amyloid beta and tau pathology are the hallmarks of sporadic Alzheimer's disease (AD) and autosomal dominant AD (ADAD). However, Lewy body pathology (LBP) is found in ≈ 50% of AD and ADAD brains. METHODS: Using an α-synuclein seed amplification assay (SAA) in cerebrospinal fluid (CSF) from asymptomatic (n = 26) and symptomatic (n = 27) ADAD mutation carriers, including 12 with known neuropathology, we investigated the timing of occurrence and prevalence of SAA positive reactivity in ADAD in vivo. RESULTS: No asymptomatic participant and only 11% (3/27) of the symptomatic patients tested SAA positive. Neuropathology revealed LBP in 10/12 cases, primarily affecting the amygdala or the olfactory areas. In the latter group, only the individual with diffuse LBP reaching the neocortex showed α-synuclein seeding activity in CSF in vivo. DISCUSSION: Results suggest that in ADAD LBP occurs later than AD pathology and often as amygdala- or olfactory-predominant LBP, for which CSF α-synuclein SAA has low sensitivity. HIGHLIGHTS: Cerebrospinal fluid (CSF) real-time quaking-induced conversion (RT-QuIC) detects misfolded α-synuclein in ≈ 10% of symptomatic autosomal dominant Alzheimer's disease (ADAD) patients. CSF RT-QuIC does not detect α-synuclein seeding activity in asymptomatic mutation carriers. Lewy body pathology (LBP) in ADAD mainly occurs as olfactory only or amygdala-predominant variants. LBP develops late in the disease course in ADAD. CSF α-synuclein RT-QuIC has low sensitivity for focal, low-burden LBP.

Improving protocols for α-synuclein seed amplification assays: analysis of preanalytical and analytical variables and identification of candidate parameters for seed quantification.

OBJECTIVES: The effect of preanalytical and analytical factors on the α-synuclein (α-syn) seed amplification assay's (SAA) performance has not been fully explored. Similarly, there is limited knowledge about the most suitable assay protocol and kinetic parameters for misfolded α-syn seed quantification. METHODS: We studied the effect of centrifugation, repeated freeze-thaw cycles (up to seven), delayed freezing, detergent addition, and blood contamination on the performance of the cerebrospinal fluid (CSF) α-syn SAA real-time quaking-induced conversion (RT-QuIC). Moreover, we analysed the inter- and intra-plate variability, the recombinant protein batch effect, and the RT-QuIC parameters' variability when multiple samples were run in controlled conditions. Finally, we evaluated the assay potential of quantifying α-syn seed by assessing kinetic curves in serial CSF dilutions. RESULTS: Among tested preanalytical variables, a ≥0.01 % blood contamination and adding detergents significantly affected the RT-QuIC kinetic parameters and the number of positive replicates. Increasing the number of replicates improved result reproducibility. The number of positive replicates in serially diluted CSF samples improved discrimination between samples with high and low seeding activity, and the time to threshold (LAG) was the most reliable kinetic parameter in multiple experiment settings. CONCLUSIONS: Preanalytical variables affecting α-syn RT-QuIC performance are limited to blood contamination and detergent addition. The number of positive replicates and the LAG are the most reliable variables for quantifying α-syn seeding activity. Their consistent measurement in serial dilution experiments, especially when associated with an increased number of sample replicates, will help to develop the α-syn RT-QuIC assay further into a quantitative test.

Performance of a seed amplification assay for misfolded alpha-synuclein in cerebrospinal fluid and brain tissue in relation to Lewy body disease stage and pathology burden.

The development of in vitro seed amplification assays (SAA) detecting misfolded alpha-synuclein (αSyn) in cerebrospinal fluid (CSF) and other tissues has provided a pathology-specific biomarker for Lewy body disease (LBD). However, αSyn SAA diagnostic performance in early pathological stages or low Lewy body (LB) pathology load has only been assessed in small cohorts. Moreover, the relationship between SAA kinetic parameters, the number of αSyn brain seeds and the LB pathology burden assessed by immunohistochemistry has never been systematically investigated. We tested 269 antemortem CSF samples and 138 serially diluted brain homogenates from patients with and without neuropathological evidence of LBD in different stages by the αSyn Real-Time Quaking-Induced Conversion (RT-QuIC) SAA. Moreover, we looked for LB pathology by αSyn immunohistochemistry in a consecutive series of 604 Creutzfeldt-Jakob disease (CJD)-affected brains. αSyn CSF RT-QuIC showed 100% sensitivity in detecting LBD in limbic and neocortical stages. The assay sensitivity was significantly lower in patients in early stages (37.5% in Braak 1 and 2, 73.3% in Braak 3) or with focal pathology (50% in amygdala-predominant). The average number of CSF RT-QuIC positive replicates significantly correlated with LBD stage. Brain homogenate RT-QuIC showed higher sensitivity than immunohistochemistry for the detection of misfolded αSyn. In the latter, the kinetic parameter lag phase (time to reach the positive threshold) strongly correlated with the αSyn seed concentration in serial dilution experiments. Finally, incidental LBD prevalence was 8% in the CJD cohort. The present results indicate that (a) CSF RT-QuIC has high specificity and sufficient sensitivity to detect all patients with LB pathology at Braak stages > 3 and most of those at stage 3; (b) brain deposition of misfolded αSyn precedes the formation of LB and Lewy neurites; (c) αSyn SAA provides "quantitative" information regarding the LB pathology burden, with the lag phase and the number of positive replicates being the most promising variables to be used in the clinical setting.

CSF α-synuclein seed amplification kinetic profiles are associated with cognitive decline in Parkinson's disease.

Seed amplification assays have been implemented in Parkinson's disease to reveal disease-specific misfolded alpha-synuclein aggregates in biospecimens. While the assays' qualitative dichotomous seeding response is valuable to stratify and enrich cohorts for alpha-synuclein pathology in general, more quantitative parameters that are associated with clinical dynamics of disease progression and that might potentially serve as exploratory outcome measures in clinical trials targeting alpha-synuclein would add important information. To evaluate whether the seeding kinetic parameters time required to reach the seeding threshold (LAG phase), the peak of fluorescence response (Imax), and the area under the curve (AUC) are associated with clinical trajectories, we analyzed LAG, Imax, and AUC in relation to the development of cognitive decline in a longitudinal cohort of 199 people with Parkinson's disease with positive CSF alpha-synuclein seeding status. Patients were stratified into tertiles based on their individual CSF alpha-synuclein seeding kinetic properties. The effect of the kinetic parameters on longitudinal development of cognitive impairment defined by MoCA ≤25 was analyzed by Cox-Regression. Patients with a higher number of positive seeding replicates and tertile groups of shorter LAG, higher Imax, and higher AUC showed a higher prevalence of and a shorter duration until cognitive impairment longitudinally (3, 6, and 3 years earlier with p ≤ 0.001, respectively). Results remained similar in separate subgroup analyses of patients with and without GBA mutation. We conclude that a more prominent alpha-synuclein seeding kinetic profile translates into a more rapid development of cognitive decline.

Kinetic parameters of alpha-synuclein seed amplification assay correlate with cognitive impairment in patients with Lewy body disorders.

The alpha-synuclein (aSyn) seed amplification assay (SAA) can identify aSyn aggregates as indicator for Lewy body pathology in biomaterials of living patients and help in diagnosing Parkinson´s disease and dementia syndromes. Our objective was to confirm that qualitative results of aSyn SAA are reproducible across laboratories and to determine whether quantitative findings correlate with patient clinical characteristics. Therefore cerebrospinal fluid samples were re-analysed by aSyn SAA in a second laboratory with four technical replicates for each sample. Kinetic parameters derived from each aggregation curve were summarized and correlated with patient characteristics. We found that qualitative findings were identical between the two laboratories for 54 of 55 patient samples. The number of positive replicates for each sample also showed good agreement between laboratories. Moreover, specific kinetic parameters of the SAA showed a strong correlation with clinical parameters, notably with cognitive performance evaluated by the Montreal Cognitive Assessment. We concluded that SAA findings are highly reproducible across laboratories following the same protocol. SAA reports not only the presence of Lewy pathology but is also associated with clinical characteristics. Thus, aSyn SAA can potentially be used for patient stratification and determining the target engagement of aSyn targeting treatments.

Clinical effects of Lewy body pathology in cognitively impaired individuals.

There is poor knowledge about the clinical effects of Lewy body (LB) pathology in patients with cognitive impairment, especially when coexisting with Alzheimer's disease (AD) pathology (amyloid-ß and tau). Using a seed amplification assay, we analyzed cerebrospinal fluid for misfolded LB-associated α-synuclein in 883 memory clinic patients with mild cognitive impairment or dementia from the BioFINDER study. Twenty-three percent had LB pathology, of which only 21% fulfilled clinical criteria of Parkinson's disease or dementia with Lewy bodies at baseline. Among these LB-positive patients, 48% had AD pathology. Fifty-four percent had AD pathology in the whole sample (17% of mild cognitive impairment and 24% of patients with dementia were also LB-positive). When examining independent cross-sectional effects, LB pathology but not amyloid-ß or tau, was associated with hallucinations and worse attention/executive, visuospatial and motor function. LB pathology was also associated with faster longitudinal decline in all examined cognitive functions, independent of amyloid-ß, tau, cognitive stage and a baseline diagnosis of dementia with Lewy bodies/Parkinson's disease. LB status provides a better precision-medicine approach to predict clinical trajectories independent of AD biomarkers and a clinical diagnosis, which could have implications for the clinical management of cognitive impairment and the design of AD and LB drug trials.

Cognitive effects of Lewy body pathology in clinically unimpaired individuals.

α-Synuclein aggregates constitute the pathology of Lewy body (LB) disease. Little is known about the effects of LB pathology in preclinical (presymptomatic) individuals, either as isolated pathology or coexisting with Alzheimer's disease (AD) pathology (ß-amyloid (Aß) and tau). We examined the effects of LB pathology using a cerebrospinal fluid α-synuclein-seed amplification assay in 1,182 cognitively and neurologically unimpaired participants from the BioFINDER study: 8% were LB positive, 26% Aß positive (13% of those were LB positive) and 16% tau positive. LB positivity occurred more often in the presence of Aß positivity but not tau positivity. LB pathology had independently negative effects on cross-sectional and longitudinal global cognition and memory and on longitudinal attention/executive function. Tau had cognitive effects of a similar magnitude, but these were less pronounced for Aß. Participants with both LB and AD (Aß and tau) pathology exhibited faster cognitive decline than those with only LB or AD pathology. LB, but not AD, pathology was associated with reduced sense of smell. Only LB-positive participants progressed to clinical LB disease over 10 years. These results are important for individualized prognosis, recruitment and choice of outcome measures in preclinical LB disease trials, but also for the design of early AD trials because >10% of individuals with preclinical AD have coexisting LB pathology.

Defining the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease MV2K: the kuru plaque type.

The current classification of sporadic Creutzfeldt-Jakob disease identifies six major subtypes mainly defined by the combination of the genotype at polymorphic codon 129 (methionine/M or valine/V) of the prion protein gene and the type (1 or 2) of misfolded prion protein accumulating in the brain (e.g. MM1, MM2, MV1, MV2, etc.). Here, we systematically characterized the clinical and histo-molecular features associated with the third prevalent subtype, the MV2 subtype with kuru plaques (MV2K), in the most extensive series collected to date. We evaluated neurological histories, cerebrospinal biomarkers, brain MRI and EEG results in 126 patients. The histo-molecular assessment included misfolded prion protein typing, standard histologic staining and immunohistochemistry for prion protein in several brain areas. We also investigated the prevalence and topographic extent of coexisting MV2-cortical features, the number of cerebellar kuru plaques and their effect on clinical phenotype. Systematic regional typing revealed a western blot profile of misfolded prion protein comprising a doublet of 19 and 20 kDa unglycosylated fragments, with the former more prominent in neocortices and the latter in the deep grey nuclei. The 20/19 kDa fragment ratio positively correlated with the number of cerebellar kuru plaques. The mean disease duration was exceedingly longer than in the typical MM1 subtype (18.0 versus 3.4 months). Disease duration correlated positively with the severity of pathologic change and the number of cerebellar kuru plaques. At the onset and early stages, patients manifested prominent, often mixed, cerebellar symptoms and memory loss, variably associated with behavioural/psychiatric and sleep disturbances. The cerebrospinal fluid prion real-time quaking-induced conversion assay was positive in 97.3% of cases, while 14-3-3 protein and total-tau positive tests were 52.6 and 75.9%. Brain diffusion-weighted MRI showed hyperintensity of the striatum, cerebral cortex and thalamus in 81.4, 49.3 and 33.8% of cases, and a typical profile in 92.2%. Mixed histotypes (MV2K + MV2-cortical) showed an abnormal cortical signal more frequently than the pure MV2K (64.7 versus 16.7%, P = 0.007). EEG revealed periodic sharp-wave complexes in only 8.7% of participants. These results further establish MV2K as the most common 'atypical' subtype of sporadic Creutzfeldt-Jakob disease, showing a clinical course that often challenges the early diagnosis. The plaque-type aggregation of the misfolded prion protein accounts for most of the atypical clinical features. Nonetheless, our data strongly suggest that the consistent use of the real-time quaking-induced conversion assay and brain diffusion-weighted MRI allows an accurate early clinical diagnosis in most patients.

Misfolded α-Synuclein Assessment in the Skin and CSF by RT-QuIC in Isolated REM Sleep Behavior Disorder.

BACKGROUND AND OBJECTIVES: Real-time quaking-induced conversion (RT-QuIC) assay detects misfolded α-synuclein (AS) in the skin and CSF of patients with the synucleinopathies Parkinson disease and dementia with Lewy bodies. Isolated REM sleep behavior disorder (IRBD) constitutes the prodromal stage of these synucleinopathies. We aimed to compare the ability of RT-QuIC to identify AS in the skin and CSF of patients with IRBD. METHODS: This was a cross-sectional study where consecutive patients with polysomnographic-confirmed IRBD and age-matched controls without RBD underwent skin biopsy and lumbar puncture the same day. Three-millimeter skin punch biopsies were obtained bilaterally in the cervical region from dorsal C7 and C8 dermatomes and in distal legs. RT-QuIC assessed AS in these 6 skin sites and the CSF. RESULTS: We recruited 91 patients with IRBD and 41 controls. In the skin, sensitivity to detect AS was 76.9% (95% CI 66.9-85.1), specificity 97.6% (95% CI 87.1-99.9) positive predictive value 98.6% (95% CI 91.0-99.8), negative predictive value 65.6% (95% CI 56.6-73.6), and accuracy 83.3% (95% CI 75.9-89.3). In the CSF, the sensitivity was 75.0% (95% CI 64.6-83.6), the specificity was 97.5% (95% CI 86.8-99.9), the positive predictive value was 98.5% (95% CI 90.5-99.8), the negative predictive value was 63.9% (95% CI 55.2-71.9), and the accuracy was 82.0% (95% CI 74.3-88.3). Results in the skin and CSF samples showed 99.2% agreement. Compared with negative patients, RT-QuIC AS-positive patients had a higher likelihood ratio of prodromal Parkinson disease (p < 0.001) and showed more frequently hyposmia (p < 0.001), dopamine transporter imaging single-photon emission CT deficit (p = 0.002), and orthostatic hypotension (p = 0.014). No severe or moderate adverse effects were reported. There was no difference between the percentage of participants reporting mild adverse events secondary to skin biopsy or lumbar puncture (9.1% vs 17.2%; p = 0.053). One hundred and ten (83%) and 104 (80%) participants, respectively, stated they would accept to undergo skin biopsy and lumbar puncture again for research purposes. DISCUSSION: Our study in IRBD shows that (1) RT-QuIC detects AS in the skin and CSF with similar high sensitivity, specificity, and agreement, (2) AS RT-QuIC positivity is associated with supportive features and biomarkers of synucleinopathy, and (3) skin punch biopsy and lumbar puncture have comparable mild adverse effects, tolerance, and acceptance. RT-QuIC in the skin or CSF might represent a patient selection strategy for future neuroprotective trials targeting AS in IRBD. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that RT-QuIC-detected AS in the skin and CSF distinguishes patients with IRBD from controls.

Evaluation of the impact of CSF prion RT-QuIC and amended criteria on the clinical diagnosis of Creutzfeldt-Jakob disease: a 10-year study in Italy.

BACKGROUND: The introduction of the prion Real-Time Quaking-Induced Conversion assay (RT-QuIC) has led to a revision of the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD).Validation studies are needed for the amended criteria, especially for their diagnostic value in the clinical setting. METHODS: We studied 1250 patients with suspected CJD referred for diagnosis to two Italian reference centres between 2010 and 2020. Focusing on the first diagnostic assessment, we compared the diagnostic value of the old and the amended criteria and that of different combinations of clinical variables and biomarker results. RESULTS: The studied cohort comprised 850 participants with CJD (297 definite sCJD, 151 genetic CJD, 402 probable sCJD) and 400 with non-CJD (61 with neuropathology). At first clinical evaluation, the sensitivity of the old criteria (76.8%) was significantly lower than that of the amended criteria (97.8%) in the definite CJD cohort with no difference between definite and probable sCJD cases. Specificity was ~94% for both criteria against the non-CJD cohort (82.0% against definite non-CJD group). Cerebrospinal fluid (CSF) RT-QuIC was highly sensitive (93.9%) and fully specific against definite non-CJD patients. Limiting the criteria to a positive RT-QuIC or/and the combination of a clinical course compatible with possible CJD with a positive MRI (Q-CM criteria) provided higher diagnostic accuracy than both the old and amended criteria, overcoming the suboptimal specificity of ancillary test results (ie, CSF protein 14-3-3). CONCLUSIONS: CSF RT-QuIC is highly sensitive and specific for diagnosing CJD in vitam. The Q-CM criteria provide a high diagnostic value for CJD.

In vivo assessment of Lewy body and beta-amyloid copathologies in idiopathic normal pressure hydrocephalus: prevalence and associations with clinical features and surgery outcome.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is a clinico-radiological syndrome of elderly individuals likely sustained by different neurodegenerative changes as copathologies. Since iNPH is a potentially reversible condition, assessing neurodegenerative pathologies in vitam through CSF biomarkers and their influence on clinical features and surgical outcome represents crucial steps. METHODS: We measured α-synuclein seeding activity related to Lewy body (LB) pathology by the real-time quaking-induced conversion assay (RT-QuIC) and Alzheimer disease core biomarkers (proteins total-tau, phospho-tau, and amyloid-beta) by immunoassays in the cerebrospinal fluid (CSF) of 293 iNPH patients from two independent cohorts. To compare the prevalence of LB copathology between iNPH participants and a control group representative of the general population, we searched for α-synuclein seeding activity in 89 age-matched individuals who died of Creutzfeldt-Jakob disease (CJD). Finally, in one of the iNPH cohorts, we also measured the CSF levels of neurofilament light chain protein (NfL) and evaluated the association between all CSF biomarkers, baseline clinical features, and surgery outcome at 6 months. RESULTS: Sixty (20.5%) iNPH patients showed α-synuclein seeding activity with no significant difference between cohorts. In contrast, the prevalence observed in CJD was only 6.7% (p = 0.002). Overall, 24.0% of iNPH participants showed an amyloid-positive (A+) status, indicating a brain co-pathology related to Aß deposition. At baseline, in the Italian cohort, α-synuclein RT-QuIC positivity was associated with higher scores on axial and upper limb rigidity (p = 0.003 and p = 0.011, respectively) and lower MMSEc scores (p = 0.003). A+ patients showed lower scores on the MMSEc (p = 0.037) than A- patients. Higher NfL levels were also associated with lower scores on the MMSEc (rho = -0.213; p = 0.021). There were no significant associations between CSF biomarkers and surgical outcome at 6 months (i.e. responders defined by decrease of 1 point on the mRankin scale). CONCLUSIONS: Prevalent LB- and AD-related neurodegenerative pathologies affect a significant proportion of iNPH patients and contribute to cognitive decline (both) and motor impairment (only LB pathology) but do not significantly influence the surgical outcome at 6 months. Their effect on the clinical benefit after surgery over a more extended period remains to be determined.

Linking the phenotype of SNCA Triplication with PET-MRI imaging pattern and alpha-synuclein CSF seeding.

Lewy-body pathology with aggregation of abnormal conformations of the protein alpha-synuclein (α-Syn) represent the histopathological hallmarks of Parkinson's disease (PD). Genetic prototypes such as PD due to mutations in the alpha-synuclein gene (SNCA) offer the opportunity to evaluate α-Syn-related profiles in patient-derived biomaterial. We identified a family with a SNCA triplication and assessed the index patient for CSF α-Syn seeding capacity and levels of total α-Syn along with other neurodegenerative CSF markers (Aß1-42, total-Tau, phospho-Tau, NFL). As no published CSF data in patients with SNCA triplication are available, we descriptively compared his CSF profiles to those of sporadic PD patients and PD patients with GBA mutations as these are also specifically associated with prominent α-Syn pathology. Additionally, skin biopsies with staining for phospho-α-Syn were done. To assess cerebral glucose metabolism and brain atrophy combined positron emission tomography and magnetic resonance imaging ([18F]FDG-PET/MRI) was performed. Age at onset was 24 years and motor impairment was accompanied by prominent non-motor symptoms with early development of dementia, depression, REM sleep behavior disorder, hyposmia, and dysautonomia. Correspondingly, PET-MRI showed hypometabolism and atrophy in frontal, temporoparietal and occipital regions. CSF levels of total α-Syn were threefold higher and RT-QuIC showed remarkable α-Syn seeding activity in all kinetic categories in the SNCATriplication patient compared to patients with GBA mutations. Our results are consistent with findings that not only mutant forms but also overexpression of the wild-type α-Syn protein lead to PD and PD dementia and show a striking CSF α-Syn seeding profile, thus substantiating the role of RT-QuIC as a specific in vivo biomarker of α-Syn brain pathology.

Sporadic Creutzfeldt-Jakob disease VM1: phenotypic and molecular characterization of a novel subtype of human prion disease.

The methionine (M)-valine (V) polymorphic codon 129 of the prion protein gene (PRNP) plays a central role in both susceptibility and phenotypic expression of sporadic Creutzfeldt-Jakob diseases (sCJD). Experimental transmissions of sCJD in humanized transgenic mice led to the isolation of five prion strains, named M1, M2C, M2T, V2, and V1, based on two major conformations of the pathological prion protein (PrPSc, type 1 and type 2), and the codon 129 genotype determining susceptibility and propagation efficiency. While the most frequent sCJD strains have been described in codon 129 homozygosis (MM1, MM2C, VV2) and heterozygosis (MV1, MV2K, and MV2C), the V1 strain has only been found in patients carrying VV. We identified six sCJD cases, 4 in Catalonia and 2 in Italy, carrying MV at PRNP codon 129 in combination with PrPSc type 1 and a new clinical and neuropathological profile reminiscent of the VV1 sCJD subtype rather than typical MM1/MV1. All patients had a relatively long duration (mean of 20.5 vs. 3.5 months of MM1/MV1 patients) and lacked electroencephalographic periodic sharp-wave complexes at diagnosis. Distinctive histopathological features included the spongiform change with vacuoles of larger size than those seen in sCJD MM1/MV1, the lesion profile with prominent cortical and striatal involvement, and the pattern of PrPSc deposition characterized by a dissociation between florid spongiform change and mild synaptic deposits associated with coarse, patch-like deposits in the cerebellar molecular layer. Western blot analysis of brain homogenates revealed a PrPSc type 1 profile with physicochemical properties reminiscent of the type 1 protein linked to the VV1 sCJD subtype. In summary, we have identified a new subtype of sCJD with distinctive clinicopathological features significantly overlapping with those of the VV1 subtype, possibly representing the missing evidence of V1 sCJD strain propagation in the 129MV host genotype.

Concordance of cerebrospinal fluid real-time quaking-induced conversion across the European Creutzfeldt-Jakob Disease Surveillance Network.

BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) real-time quaking-induced conversion (RT-QuIC) has a high degree of sensitivity and specificity for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) and this has led to its being included in revised European CJD Surveillance Network diagnostic criteria for sCJD. As CSF RT-QuIC becomes more widely established, it is crucial that the analytical performance of individual laboratories is consistent. The aim of this ring-trial was to ascertain the degree of concordance between European countries undertaking CSF RT-QuIC. METHODS: Ten identical CSF samples, seven from probable or neuropathologically confirmed sCJD and three from non-CJD cases, were sent to 13 laboratories from 11 countries for RT-QuIC analysis. A range of instrumentation and different recombinant prion protein substrates were used. Each laboratory analysed the CSF samples blinded to the diagnosis and reported the results as positive or negative. RESULTS: All 13 laboratories correctly identified five of the seven sCJD cases and the remaining two sCJD cases were identified by 92% of laboratories. Of the two sCJD cases that were not identified by all laboratories, one had a disease duration >26 months with a negative 14-3-3, whilst the remaining case had a 4-month disease duration and a positive 14-3-3. A single false positive CSF RT-QuIC result was observed in this study. CONCLUSIONS: This study shows that CSF RT-QuIC demonstrates an excellent concordance between centres, even when using a variety of instrumentation, recombinant prion protein substrates and CSF volumes. The adoption of CSF RT-QuIC by all CJD surveillance centres is recommended.

Breathing Abnormalities During Sleep and Wakefulness in Rett Syndrome: Clinical Relevance and Paradoxical Relationship With Circulating Pro-oxidant Markers.

Background: Breathing abnormalities are common in Rett syndrome (RTT), a pervasive neurodevelopmental disorder almost exclusively affecting females. RTT is linked to mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. Our aim was to assess the clinical relevance of apneas during sleep-wakefulness cycle in a population with RTT and the possible impact of apneas on circulating oxidative stress markers. Methods: Female patients with a clinical diagnosis of typical RTT (n = 66), MECP2 gene mutation, and apneas were enrolled (mean age: 12.5 years). Baseline clinical severity, arterial blood gas analysis, and red blood cell count were assessed. Breathing was monitored during the wakefulness and sleep states (average recording time: 13 ± 0.5 h) with a portable polygraphic screening device. According to prevalence of breath holdings, the population was categorized into the wakefulness apnea (WA) and sleep apnea (SA) groups, and apnea-hypopnea index (AHI) was calculated. The impact of respiratory events on oxidative stress was assessed by plasma and intra-erythrocyte non-protein-bound iron (P-NPBI and IE-NPBI, respectively), and plasma F2-isoprostane (F2-IsoP) assays. Results: Significant prevalence of obstructive apneas with values of AHI > 15 was present in 69.7% of the population with RTT. The group with SA showed significantly increased AHI values > 15 (p = 0.0032), total breath holding episodes (p = 0.007), and average SpO2 (p = 0.0001) as well as lower nadir SpO2 (p = 0.0004) compared with the patients with WAs. The subgroups of patients with WA and SA showed no significant differences in arterial blood gas analysis variables (p > 0.089). Decreased mean cell hemoglobin (MCH) (p = 0.038) was observed in the group with WAs. P-NPBI levels were significantly higher in the group with WA than in that with SAs (p = 0.0001). Stepwise multiple linear regression models showed WA being related to nadir SpO2, average SpO2, and P-NPBI (adjusted R 2 = 0.613, multiple correlation coefficient = 0.795 p < 0.0001), and P-NPBI being related to average SpO2, blood PaCO2, red blood cell mean corpuscular volume (MCV), age, and topiramate treatment (adjusted R 2 = 0.551, multiple correlation coefficient = 0.765, p < 0.0001). Conclusion: Our findings indicate that the impact of apneas in RTT is uneven according to the sleep-wakefulness cycle, and that plasma redox active iron represents a potential novel therapeutic target.

Phenotypic Heterogeneity of Variably Protease-Sensitive Prionopathy: A Report of Three Cases Carrying Different Genotypes at PRNP Codon 129.

Variably protease-sensitive prionopathy is an exceedingly rare, likely underestimated, sporadic prion disease that is characterized by heterogeneous and often non-specific clinical and pathological features posing diagnostic challenges. We report the results of a comprehensive analysis of three emblematic cases carrying different genotypes at the methionine (M)/valine (V) polymorphic codon 129 in the prion protein gene (PRNP). Clinical, biochemical, and neuropathological findings highlighted the prominent role of the host genetic background as a phenotypic modulator. In particular, the PRNP codon 129 showed a remarkable influence on the physicochemical properties of the pathological prion protein (PrPSc), especially on the sensitivity to proteinase K (PK) digestion (VV > MV > MM), which variably affected the three main fragments (i.e., of 19, 17, and 7 kDa, respectively) comprising the PrPSc profile after PK digestion and immunoblotting. This, in turn, correlated with significant differences in the ratio between the 19 kDa and the 7 kDa fragments which was highest in the MM case and lowest in the VV one. The relative amount of cerebral and cerebellar PrP mini-plaques immunohistochemistry showed a similar association with the codon 129 genotype (i.e., VV > MV > MM). Clinical manifestations and results of diagnostic investigations were non-specific, except for the detection of prion seeding activity by the real-time quaking-induced conversion assay in the only cerebrospinal fluid sample that we tested (from patient 129VV).

Neurofilament light chain and α-synuclein RT-QuIC as differential diagnostic biomarkers in parkinsonisms and related syndromes.

Neurofilament light chain (NfL) and α-synuclein oligomeric seeds (α-syn-s) are promising biomarkers for patients with parkinsonism. We assessed their performance in discriminating Parkinson disease (PD) from atypical parkinsonisms (APDs) and evaluated the association between NfL levels and clinical measures of disease severity. We measured NfL in cerebrospinal fluid (CSF) and/or plasma by immunoassays and α-syn-s in CSF by real-time quaking-induced conversion (RT-QuIC) in patients with PD (n = 153), multiple system atrophy (MSA) (n = 80), progressive supranuclear palsy/cortico-basal syndrome (PSP/CBS) (n = 58), dementia with Lewy bodies (n = 64), isolated REM-sleep behaviour disorder (n = 19), and isolated autonomic failure (n = 30). Measures of disease severity included disease duration, UPDRS-III score, Hoehn and Yahr stage, orthostatic hypotension, MMSE score, and CSF amyloid-beta profile. Both CSF NfL (cNfL) and plasma NfL (pNfL) levels were markedly elevated in APDs, and allowed differentiation with PD (vs. APDs, cNfL AUC 0.96; pNfL AUC 0.95; vs. MSA cNfL AUC 0.99; pNfL AUC 0.97; vs. PSP/CBS cNfL AUC 0.94; pNfL AUC 0.94). RT-QuIC detected α-syn-s in 91.4% of PD, but only 2.5% of APDs (all MSA). In PD/PDD, motor scales significantly correlated with cNfL levels. Although pNfL and both cNfL and α-syn-s accurately distinguished PD from APDs, the combined assessment of CSF markers provided a higher diagnostic value (PD vs. APDs AUC 0.97; vs. MSA AUC 0.97; vs. PSP/CBS AUC 0.99) than RT-QuIC alone (p = 0.047 vs. APDs; p = 0.002 vs MSA; p = 0.007 vs PSP/CBS), or cNfL alone (p = 0.011 vs. APDs; p = 0.751 vs MSA; p = 0.0001 vs. PSP/CBS). The results support the use of these assays in specialised clinics.

Association between CSF alpha-synuclein seeding activity and genetic status in Parkinson's disease and dementia with Lewy bodies.

The clinicopathological heterogeneity in Lewy-body diseases (LBD) highlights the need for pathology-driven biomarkers in-vivo. Misfolded alpha-synuclein (α-Syn) is a lead candidate based on its crucial role in disease pathophysiology. Real-time quaking-induced conversion (RT-QuIC) analysis of CSF has recently shown high sensitivity and specificity for the detection of misfolded α-Syn in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In this study we performed the CSF RT-QuIC assay in 236 PD and 49 DLB patients enriched for different genetic forms with mutations in GBA, parkin, PINK1, DJ1, and LRRK2. A subgroup of 100 PD patients was also analysed longitudinally. We correlated kinetic seeding parameters of RT-QuIC with genetic status and CSF protein levels of molecular pathways linked to α-Syn proteostasis. Overall, 85% of PD and 86% of DLB patients showed positive RT-QuIC α-Syn seeding activity. Seeding profiles were significantly associated with mutation status across the spectrum of genetic LBD. In PD patients, we detected positive α-Syn seeding in 93% of patients carrying severe GBA mutations, in 78% with LRRK2 mutations, in 59% carrying heterozygous mutations in recessive genes, and in none of those with bi-allelic mutations in recessive genes. Among PD patients, those with severe GBA mutations showed the highest seeding activity based on RT-QuIC kinetic parameters and the highest proportion of samples with 4 out of 4 positive replicates. In DLB patients, 100% with GBA mutations showed positive α-Syn seeding compared to 79% of wildtype DLB. Moreover, we found an association between α-Syn seeding activity and reduced CSF levels of proteins linked to α-Syn proteostasis, specifically lysosome-associated membrane glycoprotein 2 and neurosecretory protein VGF.These findings highlight the value of α-Syn seeding activity as an in-vivo marker of Lewy-body pathology and support its use for patient stratification in clinical trials targeting α-Syn.

Homozygous R136S mutation in PRNP gene causes inherited early onset prion disease.

BACKGROUND: More than 40 pathogenic heterozygous PRNP mutations causing inherited prion diseases have been identified to date. Recessive inherited prion disease has not been described to date. METHODS: We describe the clinical and neuropathological data of inherited early-onset prion disease caused by the rare PRNP homozygous mutation R136S. In vitro PrPSc propagation studies were performed using recombinant-adapted protein misfolding cyclic amplification technique. Brain material from two R136S homozygous patients was intracranially inoculated in TgMet129 and TgVal129 transgenic mice to assess the transmissibility of this rare inherited form of prion disease. RESULTS: The index case presented symptoms of early-onset dementia beginning at the age of 49 and died at the age of 53. Neuropathological evaluation of the proband revealed abundant multicentric PrP plaques and Western blotting revealed a ~ 8 kDa protease-resistant, unglycosylated PrPSc fragment, consistent with a Gerstmann-Sträussler-Scheinker phenotype. Her youngest sibling suffered from progressive cognitive decline, motor impairment, and myoclonus with onset in her late 30s and died at the age of 48. Genetic analysis revealed the presence of the R136S mutation in homozygosis in the two affected subjects linked to homozygous methionine at codon 129. One sibling carrying the heterozygous R136S mutation, linked to homozygous methionine at codon 129, is still asymptomatic at the age of 74. The inoculation of human brain homogenates from our index case and an independent case from a Portuguese family with the same mutation in transgenic mice expressing human PrP and in vitro propagation of PrPSc studies failed to show disease transmissibility. CONCLUSION: In conclusion, biallelic R136S substitution is a rare variant that produces inherited early-onset human prion disease with a Gerstmann-Sträussler-Scheinker neuropathological and molecular signature. Even if the R136S variant is predicted to be "probably damaging", heterozygous carriers are protected, at least from an early onset providing evidence for a potentially recessive pattern of inheritance in human prion diseases.

Diagnostic Value of the CSF α-Synuclein Real-Time Quaking-Induced Conversion Assay at the Prodromal MCI Stage of Dementia With Lewy Bodies.

OBJECTIVE: To investigate whether the CSF α-synuclein (α-syn) real-time quaking-induced conversion (RT-QuIC) assay accurately identifies patients with mild cognitive impairment (MCI) due to probable Lewy body (LB) disease. METHODS: We applied α-syn RT-QuIC to 289 CSF samples obtained from 2 independent cohorts, including 81 patients with probable MCI-LB (age 70.7 ± 6.6 years, 13.6% female, Mini-Mental State Examination [MMSE] score 26.1 ± 2.4), 120 with probable MCI due to Alzheimer disease (AD) (age 68.6 ± 7.4 years, 45.8% female, MMSE score 25.5 ± 2.8), and 30 with unspecified MCI (age 65.4 ± 9.3 years, 30.0% female, MMSE score 27.0 ± 3.0). Fifty-eight individuals with no cognitive decline or evidence of neurodegenerative disease and 121 individuals lacking brain α-syn deposits at the neuropathologic examination were used as controls. RESULTS: RT-QuIC identified patients with MCI-LB against cognitively unimpaired controls with 95% sensitivity, 97% specificity, and 96% accuracy and showed 98% specificity in neuropathologic controls. The accuracy of the test for MCI-LB was consistent between the 2 cohorts (97.3% vs 93.7%). Thirteen percent of patients with MCI-AD also had a positive test; of note, 44% of them developed 1 core or supportive clinical feature of dementia with Lewy bodies (DLB) at follow-up, suggesting an underlying LB copathology. CONCLUSIONS: These findings indicate that CSF α-syn RT-QuIC is a robust biomarker for prodromal DLB. Further studies are needed to fully explore the added value of the assay to the current research criteria for MCI-LB. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that CSF α-syn RT-QuIC accurately identifies patients with MCI-LB.