Biomarkers Analysis and Clinical Manifestations in Comorbid Creutzfeldt–Jakob Disease: A Retrospective Study in 215 Autopsy Cases

Creutzfeldt–Jakob disease (CJD), the most common human prion disorder, may occur as “pure” neurodegeneration with isolated prion deposits in the brain tissue; however, comorbid cases with different concomitant neurodegenerative diseases have been reported. This retrospective study examined correlations of clinical, neuropathological, molecular-genetic, immunological, and neuroimaging biomarkers in pure and comorbid CJD. A total of 215 patients have been diagnosed with CJD during the last ten years by the Czech National Center for Prion Disorder Surveillance. Data were collected from all patients with respect to diagnostic criteria for probable CJD, including clinical description, EEG, MRI, and CSF findings. A detailed neuropathological analysis uncovered that only 11.16% were “pure” CJD, while 62.79% had comorbid tauopathy, 20.47% had Alzheimer’s disease, 3.26% had frontotemporal lobar degeneration, and 2.33% had synucleinopathy. The comorbid subgroup analysis revealed that tauopathy was linked to putaminal hyperintensity on MRIs, and AD mainly impacted the age of onset, hippocampal atrophy on MRIs, and beta-amyloid levels in the CSF. The retrospective data analysis found a surprisingly high proportion of comorbid neuropathologies; only 11% of cases were verified as “pure” CJD, i.e., lacking hallmarks of other neurodegenerations. Comorbid neuropathologies can impact disease manifestation and can complicate the clinical diagnosis of CJD.


Introduction
Creutzfeldt-Jakob disease (CJD), the most common human prion disorder [1], is neuropathologically characterized by spongiform encephalopathy involving the subcortical grey matter of the cerebral and cerebellar cortex [2]. It can also be described as encephalopathy with protease-resistant prion protein (PrP) immunoreactivity in the form of plaques, diffuse synaptic, and a patchy/perivacuolar pattern [2]. Table 1. Summary of available epidemiological, neuropathological, immunological and genetic data in non-comorbid vs. comorbid CJD cases. The first column shows the neuropathological diagnosis of patients, second shows the total number of cases in each group, third indicate the gender distribution (female/male). In the fourth column is age range with median age (in years) and the last two columns show methionine/valine polymorphism and presence of 14-3-3 protein in cerebrospinal fluid examinated by western blot.

Diagnosis
No.

Tissue Samples
Brain tissue samples were fixed for 3-4 weeks in buffered 10% formalin. Selected tissue blocks, using a standardized protocol [11], were then embedded in paraffin using an automatic tissue processor. Five-µm-thick sections were prepared and stained with hematoxylin-eosin, Klüver-Barrera, and silver impregnation methods. Thirty-six representative blocks from standardized regions were chosen for analysis.

Immunofluorescence and Immunohistochemistry
Briefly, 5-µm-thick sections of formalin-fixed and paraffin-embedded tissue samples were deparaffinized and then incubated with primary antibodies for 20 min at room temperature. For Aβ and PrP Sc antibody staining, 96% formic acid was applied prior to the primary antibody. A second layer for light microscopy visualization, consisting of secondary horseradish peroxidase-conjugated antibody (EnVision FLEX/HRP, Dako M822, Glostrup, Denmark), was applied for 20 min at room temperature. The samples were then incubated with DAB (Substrate-Chromogen Solution, Dako K3468, Glostrup, Denmark) for 10 min to visualize the reaction. Mayer's Hematoxylin Solution was used as a counterstain.

Secondary Antibodies
Detection of immunostaining was carried out using horseradish peroxidasediaminobenzidine (see above) for immunohistochemistry and secondary antibodies conjugated with Alexa Fluor ® 488 (1:1000, donkey anti-rabbit, H + L IgG, Thermo Fischer Scientific, Waltham, ME, USA) and Alexa Fluor ® 568 (1:1000, donkey anti-mouse, H + L IgG, Thermo Fischer Scientific, Waltham, ME, USA) for immunofluorescence staining. Slides incubated with only the secondary antibody were used as specificity controls.

Light Microscopy
Samples were examined, and the results of immunohistochemical methods were classified according to currently valid neuropathological criteria for individual neurodegenerative diseases. Moreover, Alzheimer's disease was subsequently scored using the National Institute on Aging-Alzheimer's Association (NIA-AA) consensus scheme [12,13], and dementia with Lewy bodies (DLB) using the DLB consensus criteria [14] with a determination of the Braak stage [15].

Confocal Microscopy
Co-expression of pathogenic protein aggregates was imaged using a Leica TCS SP5 confocal fluorescent laser scanning microscope (Leica Microsystems Inc., Wetzlar, Germany). The HCX PL APO objective was chosen with 60× magnification and an oil immersion pinhole of 1 AU. Anti-rabbit donkey IgG secondary antibody was conjugated to Alexa Fluor ® 488 and excited at 488 nm from a 65 mW multi-line argon laser, whereas anti-mouse donkey IgG conjugated to Alexa Fluor ® 568 donkey was excited at 561 nm from a 20 mW DPSS laser.

CSF Analysis
After a single lumbar puncture and collection, CSF samples were centrifuged at 5000 RPM for 5 min and stored in polypropylene tubes at −80 • C in aliquots to avoid thawing and refreezing until the analysis.
The presence of protein 14-3-3 beta (14-3-3β) was determined using a standardized western blot protocol (adapted from Green et al. [16]) and EURO-CJD standards, with stringent control quality. Briefly, samples in doublets were separated using sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. For detection of the 14-3-3β, polyclonal antibody K-19 (1:1000, cat. #sc-629; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and after being discontinued (in 2017), monoclonal antibody B-8 (1:1000, cat. #sc-133233; Santa Cruz Biotechnology) were used. Incubation with an appropriate secondary antibody (1:1000, cat. #sc-2004; Santa Cruz Biotechnology, later with the change of primary antibody cat. #sc-516102; Santa Cruz Biotechnology) was followed by chemiluminescent detection (Pierce ECL Plus Western Blotting Substrate; cat. #32132; Thermo Scientific). A weak positive test was interpreted to mean that one sample load was positive and the other negative (the positive control was always positive). CSF levels of t-tau, p-tau, and Aβ 42 were measured during routine diagnostic testing using commercially available enzyme-linked immunoassay (ELISA) kits (INNOTEST hTAU Ag, cat. #80323/81572, INNOTEST PHOSPHO-TAU (181P), cat. #80317/81574, IN-NOTEST β-AMYLOID (1-42), cat. #80324/81576, all Innogenetics/FUJIREBIO); all testing was conducted according to the manufacturer's protocol. Although the individual values of analytes in the CSF are not entirely decisive and combinations of their ratios would be more accurate [17], the values are as follows. For t-tau, the cut-off was assessed to be 1160 pg/mL with sensitivity of 90.3 % and specificity of 90.7% (AUC: 0.926, p < 0.0001). For p-tau and Aβ 42 , the indicative normative values were determined. The values of p-tau levels > 60 pg/mL and Aβ 42 levels < 430 pg/mL were considered as abnormal. Our laboratory has extensive experience determining CSF biomarkers and successfully participates in the Alzheimer's Association's external quality control program.

Brain Tissue Analysis
Native brain tissue samples were frozen at −80 • C until analysis. The presence of PrPSc was determined using a standardized western blot protocol (adapted from Collinge et al. [18]). Briefly, brain homogenates were treated with Proteinase K (

Study Population
Our study was designed as a retrospective. We analysed data from 215 patients (n = 215), age range 40-87 years, median 66 years. No family history of CJD was retraced. We included patients with postmortem confirmed sCJD and then collected data regarding clinical presentation, biochemical analysis, EEG, and neuroimaging.
Gene analysis was performed at the level of genomic DNA, assuming an effect on the protein sequence. Only the coding part of the PRNP (NM_000311) gene and the adjacent intronic region were evaluated. Genetic analysis of genes was performed from autoptic samples of definitively confirmed cases. DNA was isolated from bone marrow (QIAamp DNA Kits).

Genetic Screen
Mutation analyses by PRNP gene sequencing were performed on genomic DNA extracted from bone marrow. The targeted gene captured all exons and the flanking intronic regions of the PRNP gene to cover the splice sites.

Clinical Data
This study was conceived as a retrospective data analysis. Medical records from different hospitals across the Czech Republic were assessed; in cases with insufficient data, the concerned hospitals were directly contacted to retrieve complete data. For this study, we focused on the presence/absence of key features mentioned by the current WHO diagnostic criteria for probable CJD, i.e., dementia, pyramidal or extrapyramidal signs, visuospatial or cerebellar dysfunction, myoclonus, and akinetic mutism (see Supplementary Material).

Magnetic Resonance Imaging
Available MRI scans were assessed independently by two investigators (J.K. and R.R.) to confirm typical MRI findings listed in the WHO diagnostic criteria for probable CJD, i.e., cortical hyperintensities (typically in the frontal and periinsular areas) and basal ganglia hyperintensities (putamen and caudate) in FLAIR and DWI sequences. DWI data included in all cases an acquisition with a b value equal to 1000. Hyperintensities were evaluated qualitatively, ADC maps were used only to confirm the restriction of the diffusion. No fixed windowing was used as it has been reported to have a lower area under the receiver operating characteristic curves when used by radiologists [19]. Moreover, we used semiquantitative scales for detecting focal atrophy in temporal areas (including the hippocampi) and parietal cortices-for this purpose, we used the MTA scale [20] (measuring mesial temporal atrophy) and the Koedam score [21] (assessing parieto-occipital atrophy). Furthermore, cerebrovascular lesions were coded using the Fazekas scale [22]. All available results are summarised in the Supplementary Material section.

Statistical METHODS
The data set was split into two groups using additional diagnoses; hypothesis testing was performed at p = 0.05. Logical explanatory variables were processed using the Fisher exact test of variable independence in 2 × 2 contingency tables to obtain significant Odds Ratios (OR). Real explanatory variables were processed using the two-sampled Wilcoxon-Mann-Whitney test of median equity. All the calculations were performed in the MATLAB 2019 Statistical Toolbox.

"Pure" CJD
Immunohistochemical methods revealed the presence of several comorbidities in neuropathological examinations. Of the 215 patients, only 24 cases (11.16%) had pure CJD, i.e., lacking any other pathological intra-or extracellular aggregates. The age range of the pure CJD cases was 40-78 years, and the median age was 60 years, which is statistically significantly younger than in the comorbid subgroups.

Comorbid CJD/AD
The cohort contained 44 comorbid cases (20.47%) of CJD/AD with possible additional co-pathology. According to the revised "ABC" classification of the National Institute on Aging-Alzheimer's Association (NIA-AA) [13], changes were classified as level "none" (1 case; 0.47%), "low" (23 cases; 10.70%), or "intermediate" (19 cases; 8.84%), no patients in the "high" category were found. The presence of tauopathies was relatively common with the Alzheimer's pathology-ARTAG was diagnosed in 10 cases (4.65%), AGD in four cases (1.86%); in one of these cases (0.47%), the criteria for ARTAG + AGD was met. The age range for this group was aged 56-85 years, and the median age was 71 years.

Comorbid CJD + Synucleinopathy
Finally, five patients (2.33%) suffered from CJD with comorbid synucleinopathy; four patients (1.86%) met the criteria for DLB, and one (0.47%) had Parkinson's disease (PD). The age ranged from 59 to 76 years, with a median age of 71 years.

CSF Analysis Results
CSF analysis was performed antemortem in about 80% of cases: 174 (80.93%) patients were tested for the presence of 14-3-3β, 168 (78.14%) patients were tested for levels of t-tau, p-tau, and Aβ 42 . For CJD, the cut-off level for t-tau was set at 1200 pg/mL [17].
Results of the 14-3-3 analysis are summarised in Table 3 (full details are available in the Supplementary Material). In all groups, 14-3-3β positivity, which is one of the diagnostic criteria for probable CJD (CDC, 2018), was less common than very high t-tau levels ( Table 3). Table 3. Numbers (n) and percentage (%) of positive, low positive, negative and unanalysed results of the presence of 14-3-3β, t-tau levels and the combined presence of 14-3-3β and t-tau protein levels in CSF. Percentage is related to the whole cohort.

MRI Results
MRIs were available in 206 cases (95.81%), and FLAIR/DWI sequences were available in 188 of these (87.44%; for detailed information, see Table 1). Typical FLAIR and DWI findings meeting the WHO diagnostic criteria for probable CJD were found as follows: cortical hyperintensities in 146 of 188 cases (77.66%), basal ganglia (caudate and putaminal) hyperintensities in 122 of 188 cases (64.89%), and both cortical plus basal ganglia hyperintensities in 109 of 188 cases (57.98%). Manifest mesial temporal atrophy (Scheltens MTA score 2) was present in 32 of 206 cases (15.53%), severe parieto-occipital atrophy (Koedam score 2) was present in 22 of 206 cases (10.68%), and potentially clinically relevant ischemic subcortical white matter lesions (Fazekas score 2 and 3) were present in 32 of 188 cases (17.02%). For detail see Figure 1.  In the first column (A,D) data from subject with pure CJD-no DWI hyperintensity in putamina (arrow) is present and MTA is 0 (read as normal). In the second column (B,E) subject with tau comorbidity with mild hippocampal atrophy (MTA 1, arrow on (E)) and DWI (B) hyperintensity in putamina (horizontal arrow), caudates (vertical arrows) and with cortical ribboning (oblique arrow). In the third column (C,F) subject with CJD and AD comorbidity is shown. A moderate hyperintensity is visible not only on DWI (not shown), but as well on FLAIR image (C). Hippocampal atrophy is well pronounced, MTA 2 (arrow from right side of the (F), pointing to the left hippocampus which manifests clear atrophy).

Confocal Microscopy Colocalization Results
Multichannel fluorescence confocal microscopy was used in comorbid CJD case examinations to monitor the colocalization of individual pathological aggregates. We devoted our previous publication [24] to the morphology of colocalizing pathological prion protein and amyloid-beta, as well as pathological tau-positive inclusions. Compound plaques with either Aβ or hyperphosphorylated tau protein (h-tau) in colocalization with PrP Sc were sparse. In contrast, PrP Sc aggregates colocalized predominantly with the non-compact (diffuse) regions of Aβ plaques, and colocalization of h-tau with PrP Sc had a dotted pattern. According to the NIA-Alzheimer's association guidelines, no association between the micromorphology of plaques and type of colocalization with polymorphism at codon 129, type of PrP Sc , and the AD ABC score was found. See  protein and amyloid-beta, as well as pathological tau-positive inclusions. Compound plaques with either Aβ or hyperphosphorylated tau protein (h-tau) in colocalization with PrP Sc were sparse. In contrast, PrP Sc aggregates colocalized predominantly with the noncompact (diffuse) regions of Aβ plaques, and colocalization of h-tau with PrP Sc had a dotted pattern. According to the NIA-Alzheimer's association guidelines, no association between the micromorphology of plaques and type of colocalization with polymorphism at codon 129, type of PrP Sc , and the AD ABC score was found. See Figures 2-4.   voted our previous publication [24] to the morphology of colocalizing pathological prion protein and amyloid-beta, as well as pathological tau-positive inclusions. Compound plaques with either Aβ or hyperphosphorylated tau protein (h-tau) in colocalization with PrP Sc were sparse. In contrast, PrP Sc aggregates colocalized predominantly with the noncompact (diffuse) regions of Aβ plaques, and colocalization of h-tau with PrP Sc had a dotted pattern. According to the NIA-Alzheimer's association guidelines, no association between the micromorphology of plaques and type of colocalization with polymorphism at codon 129, type of PrP Sc , and the AD ABC score was found. See Figures 2-4.   Figure 2. (A-C) Immunofluorescence illustration of different patterns of amyloid β (Aβ; red) and prion protein (PrP Sc ; green) colocalization in compound plaques in comorbid Alzheimer's (AD) and Creutzfeldt-Jakob diseases (CJD) cases. Primary antibodies: anti-PrP (rabbit recombinant monoclonal antibody) + anti-amyloid β-protein (mouse monoclonal antibody). The secondary antibody was conjugated with either Alexa Fluor ® 488 (anti-rabbit IgG; green) or Alexa Fluor ® 568 (anti-mouse IgG; red). Scale bar indicates 10 μm. Images come from the hippocampal region (archicortical parts).

Discussion
The main findings from comparing pure CJD with different comorbid subgroups were as follows: (1) pure CJD had a significantly lower age of onset; (2) tau comorbidity was associated with a higher probability of putaminal hyperintensities and had a lower

Discussion
The main findings from comparing pure CJD with different comorbid subgroups were as follows: (1) pure CJD had a significantly lower age of onset; (2) tau comorbidity was associated with a higher probability of putaminal hyperintensities and had a lower MTA score on MRI; (3) AD comorbidity was associated with a higher age of onset, a lower probability for developing putaminal hyperintensities on MRI, and had significantly lower beta-amyloid levels in the CSF; (4) pure CJD compared to CJD/AD had a lower age at onset and a lower MTA score on MRI; (5) comorbid CJD/tau differed from comorbid CJD/AD by having a lower age of onset, lower MTA scores, and higher beta-amyloid CSF levels, and (6) comorbid co-pathology is not dependent on codon 129 homo/heterozygosity nor the genetic background of CJD.
First, the lower age of onset in "pure" CJD cases could be explained by the absence of age-related changes in the brain tissue. In this subgroup, early-onset prion pathology with rapid disease progression probably leads to death before the development of other comorbidities.
Second, comorbid tauopathies were more likely than in other subgroups associated with putaminal DWI hyperintensities on MRI. It is known that changes in these signals correlate roughly equally with vacuolation and the amount of PrP Sc deposition (and less with astrocytic gliosis) [25]. The reason why this hypersignal is more pronounced in CJD subjects with comorbid tauopathy remains unclear since isolated tauopathies are not associated with similar MRI findings, and putaminal deposition of tau protein is not typical; however, it has been reported [26]. We can, therefore, only hypothesise that in subjects with comorbid CJD and tauopathy, the presence of tau facilitates signal increases on DWI either by tau and prion protein co-occurrence or by putaminal microstructure changes, since no micromorphological differences were visible. Lower MTA scores in the CJD/tau subgroup are even more difficult to understand, as in tau positive FTLD, pronounced frontal and temporal lobe atrophy is a frequent finding, as well as in AD, for which higher MTA scores are characteristic [27].
Third, comorbid AD was linked to a higher age of onset. Again, this is a rather surprising finding; as already discussed above, one would expect neurodegenerative comorbidities to have a significant impact relative to the destruction of brain tissue and thus lead to the earlier onset and faster disease progression. Nevertheless, CJD/AD can be viewed from different angles. Pre-existing AD development is more likely to be present at older ages, and from this point of view, it would not be unexpected that comorbid CJD/AD cases are older on average. In one of our previous publications, based on morphological findings using a multichannel fluorescent confocal microscope, we speculated that Aβ 42 might be acting as PrP Sc seeds within the brain [24] since PrP-Aβ colocalization predominates in the periphery of plaques where Aβ 42 is more abundant.
Nevertheless, it is important to emphasise that compound plaques represent only a minority of plaques since PrP and Aβ plaques tended to be, in most cases, located separately or formed "minimal compound" plaques. However, even this view of CJD/AD assumes faster progression in CJD/AD comorbid cases than in "pure" CJD, although the data obtained from our 10-year surveillance did not support such a tendency. MRI findings of putaminal sparing can be hypothesised by reduced basal ganglia involvement and fewer parkinsonian features in non-comorbid AD patients compared to pure FTLD patients. The observed low beta-amyloid levels in CSF were concordant with current biomarker findings in pure AD.
A recently published Italian study [10] showed data similar to our observations: the mean age of the CJD/AD cases (71.07 years for our cohort versus 76.1 years for the Italian patients) is strikingly higher than in pure CJD cases; no link was found between the existence of comorbid AD and disease subtype, prion strain, or PRNP genotype; in both cohorts, there were no patients in the "high" level of AD; data from both cohorts showed low Aβ levels in the CSF. There was, however, a surprising difference in the percentage of cases in which AD was reported: 61.5% of cases with CJD/AD reported in Italy compared to only 20.47% in our study.
Fourth, some of the previous findings became more obvious when we compared pure CJD to only comorbid CJD/AD. The difference in age of onset and MTA scores were larger between pure CJD and CJD/AD than when comparing pure CJD to all subgroups. The MTA scale is a widely used scoring system for assessing hippocampal atrophy in AD patients and may also monitor disease progression over time [28,29]. We thus suggest that AD could be present before prion disease in older patients.
Fifth, the role of AD on clinical manifestations and biomarkers in our cases was visible when comparing CJD/tau to CJD/AD subgroups. In line with previously discussed points, higher age of onset, higher MTA scores, and lower beta-amyloid CSF levels were key features of the impact of AD on our cohort.
Sixth, from the genetic point of view, we found no differences between codon 129 status of the PRNP gene or between genetic and sporadic CJD; in line with our pilot study, no prominent abnormalities in the deep genetic analysis of "pure" CJD cases and CJD cases in comorbidity with AD and tauopathies, respectively were seen, nor in the analysis of 15 genes related to the most important neurodegenerative diseases [30]. It has been suggested that increased expression of Syntaxin-6 (Stx6) in the basal ganglia could raise the risk of prion disease, and Stx-6 deposits have been identified as a risk factor for a 4R-tauopathy and progressive supranuclear palsy. The same study also suggests a possible role for GAL3ST1 [31] in encoding galactose-3-O-sulfotransferase 1, which affects myelin maintenance. This would be consistent with the finding that sphingolipid metabolism is disrupted early in the pathogenesis of prion disorders in mouse models [32].
Finally, compared to a study by Kovacs et al. [9] that focused on tau pathology in CJD and identified 69.3% of cases with prominent tau co-pathology and an additional 16.0% of cases with discrete non-significant tau-immunoreactive neurites, our percentage (11.16%) of "pure" cases was surprisingly low. A possible explanation for the difference in the percentage of "pure" cases by Kovacs et al. is that they did not discuss other pathological deposits. By contrast, our study revealed cases of rare comorbidities such as CJD/FTLD or CJD/synucleinopathies; some correlations between imaging findings and co-pathologies were also found.
The main limitations of our study were the single-center expertise and the very uneven extent of neuroimaging and clinical data from patients examined in different hospitals. These factors made a more robust analysis of the data and clinical correlations difficult.

Conclusions
We present results from a large cohort of postmortem confirmed CJD patients (with pure CJD and neurodegenerative comorbidities) with clinical, MRI, CSF, neuropathological, and immunohistochemical data. Our retrospective data analysis found a surprisingly high proportion of comorbid neuropathologies, with only 11% of our cases being pure non-comorbid CJD. These patients were found to have the lowest age of disease onset.
The most interesting findings from our comorbid subgroup analysis were that tauopathy is linked to putaminal hyperintensity one MRIs and that AD is associated with the age of disease onset, the degree of hippocampal atrophy seen on MRIs (low MTA scores), and the low beta-amyloid levels in the CSF. However, further investigation on a broader spectrum of comorbid neuropathologies is needed before evidence of their impact on the clinical presentation can enter routine practice, especially when new biological and potentially targeted therapies become available for treating specific proteinopathies.

Data Availability Statement:
The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscript.