medicina Review Role of Biomarkers for the Diagnosis of Prion Diseases: A Narrative Review Miren Altuna 1,2,3, * , Iñigo Ruiz 1 , María Victoria Zelaya 4 and Maite Mendioroz 5,6 1 Sant Pau Memory Unit, Hospital de la Santa Creu i Sant Pau—Biomedical Research Institute Sant Pau—Universitat Autònoma de Barcelona, 08041 Barcelona, Spain; iruizb@santpau.cat 2 Centre of Biomedical Investigation Network for Neurodegenerative Diseases (CIBERNED), 28031 Madrid, Spain 3 CITA-Alzheimer Foundation, 20009 Donostia-San Sebastián, Spain 4 Department of Pathological Anatomy, Hospital Universitario de Navarra, 31008 Pamplona, Spain; mv.zelaya.huerta@navarra.es 5 Department of Neurology, Hospital Universitario de Navarra, 31008 Pamplona, Spain; maitemendilab@gmail.com 6 Neuroepigenetics Laboratory-Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra (UPNA), IdiSNA (Navarra Institute for Health Research), 31006 Pamplona, Spain * Correspondence: maltuna@cita-alzheimer.org; Tel.: +34-935-56-59-86; Fax: +34-935-56-56-02 Abstract: Prion diseases are progressive and irreversible neurodegenerative disorders with a low incidence (1.5–2 cases per million per year). Genetic (10–15%), acquired (anecdotal) and sporadic (85%) forms of the disease have been described. The clinical spectrum of prion diseases is very varied, although the most common symptoms are rapidly progressive dementia, cerebellar ataxia and myoclonus. Mean life expectancy from the onset of symptoms is 6 months. There are currently diagnostic criteria based on clinical phenotype, as well as neuroimaging biomarkers (magnetic resonance imaging), neurophysiological tests (electroencephalogram and polysomnogram), and cerebrospinal fluid biomarkers (14-3-3 protein and real-time quaking-induced conversion (RT-QuIC)).   The sensitivity and specificity of some of these tests (electroencephalogram and 14-3-3 protein) is Citation: Altuna, M.; Ruiz, I.; Zelaya, under debate and the applicability of other tests, such as RT-QuIC, is not universal. However, the M.V.; Mendioroz, M. Role of usefulness of these biomarkers beyond the most frequent prion disease, sporadic Creutzfeldt–Jakob Biomarkers for the Diagnosis of Prion disease, remains unclear. Therefore, research is being carried out on new, more efficient cerebrospinal Diseases: A Narrative Review. fluid biomarkers (total tau, ratio total tau/phosphorylated tau and neurofilament light chain) and Medicina 2022, 58, 473. https:// potential blood biomarkers (neurofilament light chain, among others) to try to universalize access to doi.org/10.3390/medicina58040473 early diagnosis in the case of prion diseases. Academic Editor: Anastasia Bougea Keywords: prion disease; biomarkers; diagnosis; dementia; neurodegeneration Received: 19 February 2022 Accepted: 22 March 2022 Published: 25 March 2022 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Prion diseases, also known as transmissible spongiform encephalopathies, are rapidly published maps and institutional affil- progressive and irremediably fatal neurodegenerative disorders. The average life ex- iations. pectancy is six months, but great variability in the duration of the clinical course of the disease has been described, lasting from weeks to years [1–3]. The cause is the aggregation of a misfolded prion protein scrapie (PrPSc). PrPSc is the abnormal conformational isoform Copyright: © 2022 by the authors. of the normal cellular prion protein (PrPc) located on the cell surface of central nervous Licensee MDPI, Basel, Switzerland. system neurons, the exact function of which remains unknown [4]. PrPSc is able to propa- This article is an open access article gate and to aggregate in the brain tissue. PrPsc is neurotoxic and its accumulation leads to distributed under the terms and synaptic degeneration and disorganization, which induces neuronal loss and spongiform conditions of the Creative Commons changes. Indeed, a reduction of over 30% in the relative synaptic index has been reported Attribution (CC BY) license (https:// in prion disease-affected brains [1,3,5]. creativecommons.org/licenses/by/ Prion diseases are classified into sporadic (85%), genetic (10–15%) (due to mutations in 4.0/). the prion protein gene (PRNP)) and acquired (exceptional) forms (Figure 1). Prion diseases Medicina 2022, 58, 473. https://doi.org/10.3390/medicina58040473 https://www.mdpi.com/journal/medicina Medicina 2022, 58, x FOR PEER REVIEW 2 of 20 Medicina 2022, 58, 473 2 of 19 Prion diseases are classified into sporadic (85%), genetic (10–15%) (due to mutations inshow the prion protein a huge geneof(PRNP)) variety andmotor cognitive, acquired and(exceptional) forms (Figure neuropsychiatric symptoms.1). Prion dis‐ 90% of Almost eases show a huge variety of cognitive, motor and neuropsychiatric symptoms. the sporadic cases are due to sporadic Creutzfeldt–Jakob disease (sCJD) with an incidence Almost 90% of the sporadic cases are due to sporadic Creutzfeldt–Jakob disease (sCJD) with an close to 1.5–2 cases per million persons per year [3]. In addition to familial Creutzfeldt– incidence close to 1.5–2 cases per million persons per year [3]. In addition to familial Jakob disease, genetic causes include familial fatal insomnia (FFI) (of which there is also a Creutzfeldt–Jakob disease, genetic causes include familial fatal insomnia (FFI) (of which very rare sporadic form), Gerstmann–Straüssler–Scheincker (GSS) and Huntington-disease- there is also a very rare sporadic form), Gerstmann–Straüssler–Scheincker (GSS) and Hun‐ like 1 (HDL1) [6,7]. In 1996, variant Creutzfeldt–Jakob (vCJD), a zoonotic prion disease tington‐disease‐like 1 (HDL1) [6,7]. In 1996, variant Creutzfeldt–Jakob (vCJD), a zoonotic acquired by consumption of cattle contaminated by bovine spongiform encephalopathy, prion disease acquired by consumption of cattle contaminated by bovine spongiform en‐ was described cephalopathy, wasfor the firstfor described time thein thetime first United in theKingdom [8–10]. Fortunately, United Kingdom sanitary mea- [8–10]. Fortunately, sures and food chain regulation have contributed to the near disappearance sanitary measures and food chain regulation have contributed to the near disappearance of vCJD [8,9]. of vCJD [8,9]. Regardless of the type of prionopathy, early diagnosis is a challenge duegreat Regardless of the type of prionopathy, early diagnosis is a challenge due to the to phe- notypic variability, with the most frequent symptoms being rapidly progressive the great phenotypic variability, with the most frequent symptoms being rapidly progres‐ dementia, cerebellar sive dementia, ataxia and myoclonus cerebellar [3]. ataxia and myoclonus [3]. Figure 1. Prion Figure diseases 1. Prion etiologic diseases classification etiologic adapted classification by theby adapted authors [10–13].[10–13]. the authors Currently, Currently,thethe definitive diagnosis definitive of prion diagnosis diseases of prion requires diseases postmortem requires anato‐ anato- postmortem mopathological mopathological examination examinationor brain biopsy, or brain which biopsy, is notisfeasible which in clinical not feasible practice in clinical [1,3] [1,3] practice (Table 1). Postmortem neuropathological study is also necessary to classify (Table 1). Postmortem neuropathological study is also necessary to classify cases cases into the into different subtypes of sCJD. Different clinicopathological subtypes of sCJD, defined the different subtypes of sCJD. Different clinicopathological subtypes of sCJD, defined by by me‐ thionine/valine polymorphism methionine/valine at codon at polymorphism 129codon of the129 PRNP of gene and the the PRNP typeand gene (based theon the (based type size of protease‐resistant fragments) of PrPSc accumulated in the brain, have on the size of protease-resistant fragments) of PrPSc accumulated in the brain, have been been de‐ scribed [3,14]. described Despite [3,14]. the development Despite of new the development of diagnostic new diagnostictools, postmortem anato‐ anato- tools, postmortem mopathological study is the gold standard technique to confirm, or completely mopathological study is the gold standard technique to confirm, or completely rule rule out,out, the the diagnosis of a prion disease and, therefore, classifications that relate clinical and ana‐ diagnosis of a prion disease and, therefore, classifications that relate clinical and anatomical tomical changes are still relevant. The molecular subtype of sCJD is an important prog‐ changes are still relevant. The molecular subtype of sCJD is an important prognostic marker nostic marker for patient survival [3]. for patient survival [3]. In recent decades, efforts have been made to advance the use of biomarkers to allow an early diagnosis of the different prionopathies. Neurophysiological and neuroimaging biomarkers and different cerebrospinal fluid (CSF) analytes (14-3-3 protein and total tau) have been progressively incorporated into the diagnostic criteria. Special mention should be made of the real-time quaking-induced conversion (RT-QuIC) technique for the detection of prion protein in CSF, a technique with the most promising results for the accurate pre- mortem diagnosis of sCJD and other prionopathies, which is already incorporated into the latest diagnostic criteria [3] (Table 1). Despite its promising results, the RT-QuIC technique is not universally available; therefore, research into other more accessible biomarkers with potentially high diagnostic performance has continued over the last few years. Medicina 2022, 58, 473 3 of 19 Table 1. Diagnostic criteria for probable and definite categories of prion diseases. Probable Disease Definitive Disease Rapidly progressive cognitive impairment AND typical EEG (generalized periodic complexes) OR typical MRI (high signal in Progressive caudate/putamen or at neurological least two cortical regions Progressive neurological syndrome AND syndrome AND sCJD either on DWI or FLAIR) OR neuropathologically OR immunocytochemically OR positive OR positive 14-3-3 AND biochemically confirmed. CJD RT-QuIC in CSF two of the following: or other tissues. -Myoclonus -Visual or cerebellar problems -Pyramidal or extrapyramidal features -Akinetic mutism Progressive Definitive CJD Progressive neuropsychiatric AND definitive Definitive CJD neuropsychiatric disorder gCJD OR disorder AND or probable CJD OR AND pathogenic AND definite or probable pathogenic in 1st degree PRNP mutation CJD in 1st degree relative PRNP mutation relative Progressive neuropsychiatric disorder with duration of illness >6 months, no history of potential iatrogenic exposure, no evidence of gCJD and no alternative diagnosis suggested by routine investigations AND positive tonsil biopsy OR bilateral pulvinar high signal on MRI AND atypical appearance of sCJD on EEG in the early Progressive neuropsychiatric disorder AND stages AND four of the following: neuropathological confirmation (spongiform change and vCJD -Early psychiatric symptoms (depression, anxiety, apathy, extensive PrP deposition with florid plaques throughout withdrawal, delusions) the cerebrum and cerebellum) -Persistent painful sensory symptoms (frank pain or dysaesthesia) -Ataxia -Myoclonus, chorea or dystonia -Dementia Progressive cerebellar syndrome in a recipient of human Progressive cerebellar syndrome or sporadic CJD with a iCJD cadaveric-derived pituitary hormone OR sporadic CJD with a recognized exposure risk AND neuropathological recognized exposure risk confirmation Less than 8 years Cognitive impairment duration AND and/or two of the absence of following: alternative -Psychiatric symptoms etiology or Progressive neurological syndrome AND VSPr AND -Parkinsonism phenotype neuropathological confirmation -Aphasia divergence from -Ataxia atypical neurode- -Myoclonus generative dementias Organic sleep disturbances. If not yet One of the clinically apparent, a following: polysomnography has to -Loss of >10 kg be performed. during the last 6 At least two of the months following: -Vegetative signs -Psychiatric symptoms (hyperhidrosis, Progressive neurological syndrome AND sFI and FFI (visual hallucinations, AND newly diagnosed neuropathological confirmation personality changes, arterial depression, anxiety, hypertonia, aggressiveness, tachycardia, disinhibition, listlessness) constipation, -Ataxia hyperthermia) -Visual symptoms -Husky voice -Myoclonus -Cognitive/mnesic deficits Progressive neurological syndrome AND Progressive cerebellar syndrome, cognitive impairment and/or neuropathological confirmation (amyloid deposits GSS sensory symptoms AND pathogenic PRNP mutation immunoreactive for PrP are the morphological hallmark of GSS) Abnormal involuntary movements, coordination difficulty, dementia, Kuru and multicentric plaques that stain with anti-prion HDL-1 personality changes and psychiatric symptoms AND pathogenic antibodies PRNP mutation CJD: Creutzfeldt-Jakob disease; sCJD: sporadic Creutzfeldt-Jakob disease; EEG: electroencephalogram; MRI: mag- netic resonance imaging; RT-QuIC: Real-Time Quacking-Induced conversion (RT-QuIC); CSF: cerebrospinal fluid; gCJD: genetic Creutzfeldt-Jakob disease; vCJD: variant Creutzfeldt-Jakob disease; iCJD: iatrogenic Creutzfeldt- Jakob disease; VSPr: Variably protease-sensitive prionopathy; sFI: sporadic fatal insomnia; FFI: fatal familial insomnia; GSS: Gerstmann-Sträusller-Scheinker; PrP: prion protein; HDL-1: Huntington disease-like syndrome. Bold words are for emphasis. Medicina 2022, 58, 473 4 of 19 This narrative review aims to examine the currently accepted neurophysiological, neuroimaging and cerebrospinal fluid (CSF) biomarkers for diagnosis and to investigate al- ternative CSF and peripheral blood biomarkers that have been recently proposed (Figure 2). We highlight those biomarkers that are more easily accessible, including blood biomarkers, which would truly represent a diagnostic revolution in prionopathies. The existing differ- ences in biomarker performance between different prionopathies will also be emphasized, although it is true that most of the work reviewed has focused on sCJD. The examination, in combination, of current diagnostic criteria and of multimodal diagnostic biomarkers (e.g., neurophysiological, neuroimaging, genetic, CSF and plasma analytes), available both for use in clinical practice and in the development phase, not only limited to CJD5but Medicina 2022, 58, x FOR PEER REVIEW of 20also to the rest of the prionopathies, is considered of special interest in this review. Figure2.2.Diagnostic Figure Diagnostic biomarkers forprion biomarkers for priondiseases diseasesininuse useinin clinical clinical practice practice andand summary summary of the of the mostpromising most promisingbiomarkers biomarkers under under investigation. investigation. Figure Figurecreated createdwith withbiorender.com. biorender.comCJD: Creutz‐ (accessed on 18 feldt–Jakob February diseases, 2022). sCJD: sporadic Creutzfeldt–Jakob CJD: Creutzfeldt–Jakob diseases, sCJD:disease; sporadic gCJD: genetic Creutzfeldt–Jakob Creutzfeldt–Jakob disease; gCJD: disease, FFI: fatal familial insomnia, sFI: sporadic fatal insomnia. genetic Creutzfeldt–Jakob disease, FFI: fatal familial insomnia, sFI: sporadic fatal insomnia. 2. Neurophysiological Biomarkers 2.1. Electroencephalogram (EEG) Periodic sharp‐wave complexes (PSWCs), generalized and/or lateralized complexes, characterized by strictly periodic cerebral potentials at a frequency of 1 Hz, are the typical Medicina 2022, 58, 473 5 of 19 2. Neurophysiological Biomarkers 2.1. Electroencephalogram (EEG) Periodic sharp-wave complexes (PSWCs), generalized and/or lateralized complexes, characterized by strictly periodic cerebral potentials at a frequency of 1 Hz, are the typical EEG finding commonly associated with sCJD. PSWCs tend to disappear during sleep and may be attenuated by some psychotropic drugs [15]. PSWCs are observed in 67–95% of patients with sCJD, with a sensitivity of 65% and specificity of 90% [16,17]. In turn, the main background frequency and the α/θ power ratio in quantitative EEG seems to be related to clinical progression and has been suggested as a useful tool for follow-up monitoring in prion diseases [18]. EEG could also be useful for the detection of non-convulsive status epilepticus, which, although infrequent, has an increased risk in some prionopathies, such as sCJD [3]. Advantages: EEG is an economically accessible and safe technique. It is available in most healthcare centers and can be performed repeatedly during clinical follow-up if required. Limitations: The diagnosis performance of EEG improves with the clinical course of the disease. Therefore, it is a biomarker that is mainly related to the late symptomatic phases of sCJD [13,15]. In early stages of the disease, unspecific alterations, such as diffuse slowing and frontal rhythmic delta activity (FIRDA), are more frequent [15]. Among the other limitations, it should be noted that similar alterations have been described in other neurodegenerative dementias, such as Alzheimer’s disease (AD) and Lewy body dementia (LBD), although less frequently. It is also noteworthy that the probability of detection of PSWC is much lower in other prionopathies, such as FFI or GSS, and even in the MV2, VV2 and MM2 forms of sCJD [3,17,19]. 2.2. Polysomnogram (PSG) In cases of familial or sporadic fatal insomnia the demonstration of an early and progressive reduction in total sleep time, the loss of sleep spindles and K-complexes, the disruption of normal sleep structure, sleep fragmentation, and periods of subwakefulness interrupted by brief episodes of REM sleep, with or without atonia, often associated with dream enactment behavior, is a diagnostic criterion [20]. In the case of sCJD, sleep anomalies are not recognized as a diagnostic criterion, although it is common to detect loss of normal sleep EEG architecture, sleep-disordered breathing [21] and periodic leg movement disorders in the PSG [22]. Advantages: A non-invasive technique with high diagnostic performance for both sporadic and familial fatal insomnia. Limitations: Not universally accessible and not useful for differential diagnosis of the most frequent causes of rapidly progressive neurodegenerative dementias apart from fatal insomnia. 3. Neuroimaging Biomarkers 3.1. Brain Magnetic Resonance (MR) The presence of hyperintensities in T2 and fluid-attenuated inversion recovery imag- ing (FLAIR) sequences are frequent in the MR images of sCJD specially involving basal ganglia [16], as well as the restriction of diffusion (DWI) in at least two cortical regions (rib- boning) or/and restricted diffusion predominantly in the caudate nucleus, putamen and/or thalamus [3,17,23,24]. However, these typical signs on MR images are not pathognomonic of sCJD and could also be induced, though rarely, by toxic metabolic encephalopathies, progressive multifocal dementia, autoimmune encephalitis, CNS lymphoma, vasculitis and infectious etiologies [24]. The MR pattern could also be useful, mainly when combined with PRNP polymor- phisms, to differentiate sCJD molecular subtypes [17]. In the MM1 subtype of sCJD, the caudate nucleus has unilateral or bilateral asymmetrical involvement, and the involvement of the thalamus is more frequent in VV2 and MV2 subtypes [25]. The presence of a pulvinar Medicina 2022, 58, 473 6 of 19 sign (high signal on FLAIR and DWI) is highly suggestive of vCJD [3,26]. Moreover, altered diffusion in the striatum, thalamus and frontal and occipital cortices has been reported in GSS [6]. The sensitivity of sCJD diagnosis using MR varies according to different stud- ies between 80 and 92%; the same is true for specificity, with a range between 74 and 98% [3,27,28]. Recently, the use of MR spectroscopy to determine the N-acetylaspartate (NAA)/creatine (Cr) ratio has been suggested as a useful parameter for predicting the clinical course in sCJD, as lower NAA/Cr is related to shorter disease duration [29]. Advantages: Structural neuroimaging is a mandatory test for the differential diagnosis of cognitive impairment, and, in the case of rapidly progressive dementias, the MR evalua- tion is crucial. Changes in restriction occur early in the context of sCJD [3,30]. In contrast with EEG, MR diffusion abnormalities are an early phenomenon, being detectable at least one year before the onset of symptoms in asymptomatic PRNP mutation carriers [31], making it useful for early diagnosis. Limitations: At the time to perform the MR, a clinical suspicion of possible prion disease must be reported because is advisable to perform an MR evaluation with a specific protocol including diffusion sequences (DWI/ADC). 3.2. Fluorodesoxyglucose Positron Emission Tomography (PET-FDG) Decreased glucose metabolism in the neocortex affecting extensive cortical regions (frontal, parietal and occipital cortices) and basal ganglia has been reported in sCJD but does not seem to be useful for differential diagnosis with other neurodegenerative dementias [28]. However, the hypometabolism of medial temporal area seems to be significantly less frequent compared to other neurodegenerative dementias [32]. Instead, it may be useful for the diagnosis of infrequent forms of sporadic fatal insomnia, where hypometabolism in the thalamic region is early and characteristic [3,6]. Advantages: It could be useful for helping with the diagnosis of sporadic fatal insomnia. Limitations: It is an expensive test that is not usually performed in cognitive de- cline screening. There are no specific hypometabolism patterns that could be useful for diagnosing sCJD or performing a differential diagnosis with other prionopathies. 4. Genetics PRNP gene sequencing is the primary diagnostic technique in genetic prion disease. PRNP mutations account for 10–15% of all human prion syndromes [3]. The detection of PRNP gene mutations can be performed by sequencing DNA from patient blood specimens or a decedent’s unfixed autopsy tissue. All the genetic forms of prion disease are linked to PRNP mutations and include point mutations, octapeptide repeat insertions and deletions. Many different mutations have been linked to genetic CJD, although the most common worldwide is E200K [2]. The penetrance of PRNP mutations is assumed to be close to 100% although real-life data are lacking. In the specific case of the E200K mutation, the most widespread significant variability has been detected with penetrance ranging from 60 to 90% among different populations [2]. The D178N mutation is present in all families with FFI. In individuals of European ancestry, five variants account for up to 85% of the pathogenic PRNP variants (Table 2). Therefore, the first step in genetic analysis is to determine whether these variants exist, and, if not, the entire gene should be sequenced [2]. However, in specific geographic regions with higher prevalence of gCJD, an adaptation of the preliminary genetic analysis could be advisable. Medicina 2022, 58, 473 7 of 19 Table 2. Summary of the most frequent PRNP variants. Adapted from Ladogana et al., 2018 [2]. PRNP DNA Nucleotide Predicted Protein Related Expected Survival Phenotype Variants Change Change Prionopathy (Median) Ataxia (100%), pyramidal (75%), dementia (62%), Proline to leucine extrapyramidal (50%), P102L c.305C>T substitution at GSS 40 months. myoclonus (25%). codon 102 Others: dysarthria, sleep and sensory disturbances. Dementia (96%), FFI and genetic myoclonus (89%), ataxia 15 months (earlier CJD aspartic acid to (82%), extrapyramidal onset and shorter (Depends on the asparagine (82%), pyramidal (79%), duration of D178N c.532G>A allele on codon 129. substitution at cortical blindness (79%). symptomatic M allele: FFI, and codon 178 Others: sleep disturbances, disease in genetic V allele: genetic dysarthria, weight loss CJD). CJD) and hyperhidrosis. Dementia (100%), extrapyramidal (54%), pyramidal (46%). Valine to isoleucine Others: akinetic mutism 16,4 months (wide V180I c.538G>A change at codon Genetic CJD (57%) and psychiatric range of survival). 180 (50%). Cortical blindness, myoclonus and ataxia are more infrequent. Ataxia (100%), dementia (95%), myoclonus (85%), pyramidal (70%), cortical Glutamic acid to 5 months (wide blindness (70%), E200K c.598G>A lysine substitution Genetic CJD range of survival extrapyramidal (65%). at codon 200 1–74 months). Others: dysarthria, sleep disturbances and weight loss. Ataxia (100%), dementia (92%), myoclonus (92%), Valine to isoleucine extrapyramidal (92%), 4 months (wide V210I c.628G>A substitution at Genetic CJD cortical blindness (85%), range of survival). codon 210 pyramidal (72%). Others: dysarthria and sensory symptoms. GSS: Gertsmann-Sträusller-Scheinker; FFI: Familial fatal insomnia; CJD: Creutzfeldt-Jakob disease; M: methionine; V: valine. Bold words are for emphasis. In addition, the polymorphism of codon 129 of the PRNP gene has potential relevance as it may influence susceptibility to both variant and sporadic forms of CJD: 85–95% of sCJD cases are methionine homozygous at codon 129, compared to 49% in healthy controls [2,17]. However, the codon 129 polymorphism has primarily been investigated in research studies and is not currently used in the diagnostic work-up of prion disease. Interestingly, stratifying patients by codon 129 polymorphism could have a possible role in future clinical trials [17]. 5. Cerebrospinal Fluid (CSF) Biomarkers 5.1. Biochemical Analysis No differences in the quantity of proteins, glucose concentration and total cell number assessed in CSF are detected in prion diseases compared to controls [33]. Advantages: The existence of significant biochemical alterations in CSF may help to check for other etiologies, e.g., inflammatory. Medicina 2022, 58, x FOR PEER REVIEW 9 of 20 Medicina 2022, 58, 473 8 of 19 Advantages: The existence of significant biochemical alterations in CSF may help to check for other etiologies, e.g., inflammatory. Limitations: Limitations: Lumbar punctureisisconsidered Lumbar puncture consideredanan invasive invasive test, test, even even though though it is itcur‐ is cur- rently performed in clinical practice for the early diagnosis of neurodegenerative disorders, rently performed in clinical practice for the early diagnosis of neurodegenerative disor‐ such ders,assuch AD.as AD. 5.2. 5.2.CSF CSFSurrogate Surrogate Biomarkers Biomarkers 5.2.1. 14-3-3 Protein 5.2.1. 14‐3‐3 Protein The Thegamma-isoform gamma‐isoform of the14‐3‐3 of the 14-3-3protein protein(14‐3‐3 (14-3-3 gamma) gamma) expressed expressed in neurons in neurons couldcould bebea aspecific specific marker for neuronal damage and is useful for sCJD diagnosis [34–36]. TheThe marker for neuronal damage and is useful for sCJD diagnosis [34–36]. pathological pathological mechanisms mechanisms leadingleadingtotothe theaccumulation accumulation of of 14-3-3 14‐3‐3 protein protein in CSF in CSF are are not not fully fully understood; however, neuronal loss followed by cell lysis is assumed to cause in‐ in- understood; however, neuronal loss followed by cell lysis is assumed to cause crease creaseinin14-3-3 14‐3‐3 levels levels [37]. Currently,the [37]. Currently, thedetection detectionofof increased increased 14-3-3 14‐3‐3 protein protein in CSF in CSF is is used usedasasaamolecular molecular diagnostic criterionfor diagnostic criterion forpatients patientsthat that areare clinically clinically compatible compatible withwith sCJD sCJD[37]. [37].The Thediagnosis performanceisislower diagnosis performance lowerforforgenetic genetic forms forms of prion of prion diseases diseases andandfor for sporadic sporadicfatalfatalinsomnia insomnia[38,39]. [38,39].TheThediagnostic diagnostic performance performance of of quantitative quantitative enzyme-linked enzyme‐ immunosorbent linked immunosorbent (ELISA)(ELISA) assay for 14-3-3 assay is higher for 14‐3‐3 in comparison is higher in comparisonwith western with westernblotting blotting (WB) (WB)Combination [34,40]. [34,40]. Combination with other withsurrogate other surrogate biomarkers, biomarkers, such assuch theasdetermination the determi‐ of nation total tauof(t-tau) total tau and(t‐tau) andof the ratio the ratio of t‐tau/phosphorylated t-tau/phosphorylated tau (p‐tau), tau (p-tau), significantly significantly increases increases the the specificity specificity [14,36,41–43]. [14,36,41–43]. Advantages: ItIt has Advantages: has high high sensitivity sensitivity (86–97%) (86–97%) forfor sCJD sCJD [44–46] [44–46] (Figure (Figure 3). 3). Combina‐ Combination tion with t‐tau and the ratio of t‐tau/p‐tau has very good sensitivity with t-tau and the ratio of t-tau/p-tau has very good sensitivity and specificity and and specificity and is is more more accessible compared to other techniques, accessible compared to other techniques, such as RT-QuIC. such as RT‐QuIC. Figure3.3. Biomarkers Figure Biomarkers used usedin in clinical practice clinical and their practice and diagnostic sensitivity their diagnostic for prionfor sensitivity diseases. prionsCJD: diseases. sporadic sCJD: Creutzfeldt–Jakob sporadic disease; Creutzfeldt–Jakob gCJD: disease; genetic gCJD: Creutzfeldt–Jakob genetic disease; Creutzfeldt–Jakob iCJD:iCJD: disease; iatrogenic iatrogenic Creutzfeldt–Jakob disease; vCJD: variant Creutzfeldt–Jakob disease; FFI: fatal familial insomnia; Creutzfeldt–Jakob disease; vCJD: variant Creutzfeldt–Jakob disease; FFI: fatal familial insomnia; GSS: GSS: Gerstmann–Sträusller–Scheinker; VPSPr: variably protease‐sensitive prionopathy; MRI: Gerstmann–Sträusller–Scheinker; VPSPr: variably protease-sensitive prionopathy; MRI: magnetic magnetic resonance imaging; EEG: electroencephalogram. resonance imaging; EEG: electroencephalogram. Limitations: Protein 14‐3‐3 is not as specific as was initially thought [16]; specificity Limitations: could Protein vary between 75.6 14-3-3 and 91%is for not sCJD as specific as was [42]. Acute initially thought neurological [16];such conditions, specificity as could vary between 75.6 and 91% for sCJD [42]. Acute neurological conditions, such as stroke, status epilepticus or inflammatory encephalopathies, can also increase 14-3-3 protein levels [47]. In turn, the approach shows poor performance in the diagnosis of infrequent prion diseases. Medicina 2022, 58, 473 9 of 19 5.2.2. Total Tau (t-tau) and Total Tau/Phosphorylated Tau (t-tau/p-tau) Ratio Elevation of CSF t-tau levels is correlated with axonal neurodegeneration rate in many different neurological conditions, while p-tau is increased in AD but not in other neurode- generative disorders. The determination of levels of t-tau and the ratio of t-tau/p-tau could be useful for the diagnosis of CJD, preferably combined with other diagnostic tools [16,48]. Sensitivity and specificity vary depending on the cut-off point established for both t-tau and t-tau/p-tau ratio values but very high sensitivity (85%) and specificity (98.6%) can be achieved, both of which are higher compared to the 14-3-3 protein performance [16,49–53]. In turn, the most highly elevated levels of t-tau are observed in the MM1, MV1 and VV2 types with classical symptomatology [54]. Therefore, it has been suggested that t-tau can be used in the diagnostic assessment of prion protein type when the codon 129 genotype is known and could provide valuable information for physicians about the prognosis [55]. Increase in t-tau and t-tau/p-tau is related to a shortened life expectancy, which could be explained because t-tau reflects neuronal damage [1,48,54,56] and t-tau levels continue to increase during the progression of the disease [57]. Levels of t-tau correlate with disease burden as assessed by cortical involvement evaluated by DWI sequence of MR [58]. Advantages: High specificity for t-tau and t-tau/p-tau ratio for sCJD. Diagnostic performance improves for both genetic and sporadic CJD if combined with 14-3-3 pro- tein [43,59,60] Limitations: t-tau could be increased in other neurodegenerative conditions but not as much as in prionopathies. 5.2.3. Neurofilament Light Chain Protein (NfL) NfL is a neuronal cytoskeleton component and is released when there is neuronal damage in a wide range of conditions, making this a very good biomarker of neurode- generation [61,62]. NfL is increased in all sCJD subtypes, including those which typically show low values of t-tau and negative protein 14-3-3 (e.g., sCJD MV2K, MM2C and gCJD E200K) [5,63]. It has outstanding sensitivity to detect sCJD, higher than 95%, but a very low specificity of 43.1% [14,64]. Limitations: Low specificity makes it less useful compared to the determination of t-tau or the combination of t-tau or ratio t-tau/p-tau + 14-3-3 protein in the differential diagnosis of rapidly progressive dementias [14]. Advantages: Due to high sensitivity, this could be useful combined with other surro- gate biomarkers for the early diagnosis of prionopathies. 5.2.4. Other Biomarkers Alpha synuclein, a neuronal protein especially abundant at presynaptic regions, stands out among other promising surrogate biomarkers. Its levels are increased in both genetic and sporadic forms of CJD, with good diagnostic performance (sensitivity 98% and specificity 97%); in addition, there is an inverse correlation between alpha synuclein levels and disease duration in CJD [61,65–68]. In turn, neurogranin, related to synaptic plasticity, has been shown to be increased in sCJD compared to controls and other neu- rodegenerative dementias with a diagnostic yield similar to 14-3-3 protein in the early stages of the disease, without significant variations with disease progression [5,10]. It has been speculated that neurogranin levels could be useful to differentiate between different subtypes of CJD (different concentrations having been reported according to the clinico- pathological subtypes) [5]. Interestingly, it has been reported that ubiquitin, which marks neuritic damage, dysfunctional protostasis and neuroinflammation, has higher concentra- tions in CJD than in controls and other neurodegenerative dementias, and especially in less frequent forms of sCJD, such as MM(V)1 [69,70]. One study has also shown higher levels of calmodulin, a ubiquitous calcium-binding protein, in sCJD compared to other neurodegenerative dementias, particularly in those with higher levels of t-tau [71]. Markers related to oxidative stress have also been postulated, as is the case for mito- chondrial malate dehydrogenase 1 (MDH1). Increase in MDH1 would have a sensitivity Medicina 2022, 58, 473 10 of 19 of 97.5% and specificity of 95.6% for the diagnosis of sCJD with positive correlation for t-tau and 14-3-3 protein concentrations according to preliminary data from several studies with small or very heterogeneous samples [72,73]; however, this must be confirmed in future studies prior to its future possible use in clinical practice. A single study indicates that glial biomarkers, such as YKL-40, CHIT-1 and GFAP, are significantly increased in the VV2 form of CJD compared to other neurodegenerative dementias and are positively correlated with symptomatic progression of the disease [10,74]. An increase in these biomarkers has even been observed in presymptomatic cases of GSS [74]. Biomarkers related to iron metabolism, such as transferrin, have also been studied. A single study has postulated that elevated transferrin could be used in combination with t-tau to increase its diagnostic performance [75]. Advantages: The discovery of new biomarkers related to different pathophysiological processes can help us to better understand the disease itself and thus identify possible therapeutic targets. Limitations: At present, none of these biomarkers are approved for use, either in isolation, or in combination with core biomarkers, in neurogenerative diseases. Further studies are needed to confirm the benefit of their application in clinical practice. 5.3. CSF Prion Proteins 5.3.1. Total PrP Total PrP levels in the CSF of patients with prion disease tend to be reduced compared to controls. It has been speculated that this may result from the sequestration of soluble monomeric protein into aggregates in the brain (analogous to the proposed mechanism for reduction of CSF Aβ1-42 in AD) [65,76,77]. Specificity for diagnosing prionopathies of reduced total PrP in CSF seems to be moderate and unlikely to be used for differential diagnosis with other causes of rapidly progressive dementia, at least if not combined with surrogate CSF biomarkers, such as 14-3-3 protein and t-tau or ratio of t-tau/p-tau [37,61,78]. On the other hand, it has been suggested that it could have potential applicability for monitoring response to future disease-modifying treatments because there are significant differences in genetic forms of CJD between PRNP mutation carriers and non-carriers years before symptom onset, and levels of test-retest of total PrP are stable during follow- up [79,80]. Advantages: This biomarker directly reflects the pathophysiology of prion diseases and therefore has a potential role in the follow-up of possible future treatments. Limitations: It has suboptimal performance, no better than that described for surrogate biomarkers with applications in clinical practice. Therefore, it is unlikely to be indicated for use as a diagnostic biomarker in the future. 5.3.2. Prion Real-Time Quaking-Induced Conversion (RT-QuIC) Aside from brain biopsy, RT-QuIC, an ultrasensitive in vitro PrPSc amplification assay (Figure 4), is the only disease-specific antemortem diagnostic biomarker that directly detects the pathological prion protein (PrPSc) and since 2018 has been incorporated in the current diagnostic criteria [17,41,42,62,81–83]. A very high sensitivity (80–96%) and virtually full specificity (99–100%) has been reported for RT-QuIC for sCJD [13] (Figure 3). However, the sensitivity is lower in MM1, the most frequent subtype of sCJD [41], and is even lower in genetic forms of CJD, GSS and FFI, as well as in sporadic fatal insomnia. The most plausible explanation for this finding is strain variability [76,81]. Nevertheless, the RT-QuIC performance continues to be higher compared to 14-3-3 protein and to the combination of 14-3-3 protein and t-tau or t-tau/p-tau ratio for genetic forms of CJD, GSS and FFI [77]. However, it has not demonstrated an ability to differentiate between different subtypes of sCJD [84]. In turn, RT-QuIC presents increased costs and less inter-laboratory standardization data is available compared to the use of approved surrogate biomarkers (14-3-3 protein and t-tau) [85]. The most plausible explanation for this finding is strain variability [76,81]. Nevertheless the RT‐QuIC performance continues to be higher compared to 14‐3‐3 protein and to the combination of 14‐3‐3 protein and t‐tau or t‐tau/p‐tau ratio for genetic forms of CJD, GSS and FFI [77]. However, it has not demonstrated an ability to differentiate between differ ent subtypes of sCJD [84]. In turn, RT‐QuIC presents increased costs and less inter‐labor Medicina 2022, 58, 473 11 of 19 atory standardization data is available compared to the use of approved surrogate bi omarkers (14‐3‐3 protein and t‐tau) [85]. Figureof Figure 4. Description 4. the Description of the prion prion real-time real‐time quaking‐induced quaking-induced conversionapplied conversion technique technique applied to th to the diagnosis of prion diseases, adapted from Orrù et al., 2017 [86]. diagnosis of prion diseases, adapted from Orrù et al., 2017 [86]. Advantages: TheAdvantages: ability to The ability detect priontoprotein detect prion protein with high with high sensitivity sensitivity and andofspecificity specificity of almost 100%. Included as a diagnostic almost 100%. Included as a diagnostic criterion. criterion. Limitations:more Limitations: Technically Technically complexmore complex than than the determination the determination of otherofsurrogate other surrogate bi omarkers that are measurable in CSF (14‐3‐3 protein and total tau), and accessible in fewe biomarkers that are measurable in CSF (14-3-3 protein and total tau), and accessible in hospitals. Poorer performance for atypical variants of sCJD and genetic forms of CJD, GSS fewer hospitals. Poorer performance for atypical variants of sCJD and genetic forms of CJD, and familial and/or sporadic fatal insomnia. GSS and familial and/or sporadic fatal insomnia. 6. Plasma Biomarkers 6. Plasma Biomarkers 6.1. NfL 6.1. NfL Increased levels Increased of plasmalevels NfLofhave plasma beenNfL have been reported reported in CJD, in CJD, significantly significantly higher than in higher than other neurodegenerative dementias (e.g., AD, LBD and frontotemporal dementia -FTD-) ‐FTD in other neurodegenerative dementias (e.g., AD, LBD and frontotemporal dementia ) and obviously compared to controls. One study has shown an AUC of 0.93 to discrimi and obviously compared to controls. One study has shown an AUC of 0.93 to discriminate nate CJD from non‐CJD dementias [87] and another has reported a sensitivity of 100% and CJD from non-CJD dementias [87] and another has reported a sensitivity of 100% and specificity of 85.5% for the diagnosis of different prionopathies (sporadic, genetic and iat specificity of 85.5% for the diagnosis of different prionopathies (sporadic, genetic and rogenic CJD and GSS) [61,88]. However, it has been suggested that performance of NfL i iatrogenic CJD and GSS) [61,88]. However, it has been suggested that performance of better in CSF compared to plasma [89]. The increase in plasma NfL occurs in the early NfL is better instages CSF ofcompared to plasma [89]. The increase in plasma NfL occurs in the the disease, before symptom onset, and it seems to be always altered if the prion early stages of conversion the disease,assay before symptom is positive onset, in CSF. and The it seems levels of NfLtocontinue be always altered if to increase thethe symp with prion conversion assay is positive in CSF. The levels tomatic progression of the disease [88,90,91].of NfL continue to increase with the symptomatic progression of theAn Advantages: disease [88,90,91]. accessible biomarker with a huge number of studies suggesting it Advantages: Anfor utility accessible biomarker discriminating with a huge number neurodegenerative of studies suggesting vs. non‐neurodegenerative its dementias. Al utility for discriminating tered from neurodegenerative vs. non-neurodegenerative the early stages of disease, so therefore useful fordementias. Altered early diagnosis. from the early stages of disease, so therefore useful for early diagnosis. Limitations: Not a specific biomarker of prion diseases; also increased in other neu- rodegenerative dementias, although not to the same extent. 6.2. t-tau Plasma t-tau levels are higher in CJD, in sporadic, iatrogenic and genetic forms, com- pared not only to controls, but also significantly when compared to other neurodegenerative dementias [25,39,41]. t-tau is particularly increased in sCJD patients who are homozygous for methionine at codon 129 of the PRNP gene [92]. Plasma t-tau seems to have a moderate association with disease duration, offering a moderate survival prediction capacity [93]. One study has reported a sensitivity of 84.6% and specificity of 96.2% [88], but it seems that both sensitivity and specificity are lower in general for plasma t-tau compared to CSF t-tau [89]. Medicina 2022, 58, 473 12 of 19 Advantages: This could be useful to discriminate from non-neurodegenerative causes of cognitive decline. Limitations: It is a non-specific biomarker of neurodegeneration that does not clearly improve performance compared to use of plasma NfL. 6.3. YKL-40 Increased levels of plasma YKL-40 have been detected in different sporadic and genetic forms of prionopathies compared to controls. Higher levels are detected in later stages of the disease [94]. Due to the limited capacity to discriminate from other neurodegenerative dementias and moderate ability to distinguish from healthy controls, YKL-40 does not seem to be a good diagnostic biomarker but could have a role in disease monitoring [94]. Advantages: Could be a potential plasma biomarker related to progression of the disease. Limitations: Very limited capacity to discriminate prion diseases from other neurode- generative diseases; doubtful utility as an additional biomarker for diagnosis. 6.4. MicroRNA The blood microRNA profile has been suggested as a complementary test to use, together with plasma neurodegeneration biomarkers. One study, with a small sample size, has shown different microRNA expression in sCJD compared not only to controls but also to AD [95]. Advantages: Could be another non-invasive biomarker of sCJD. Limitations: Very limited data; does not seem to be applicable in clinical practice, at least in the short term. 6.5. Total Prion Protein (t-PrP) The existence of vascular pathology with loss of integrity of the blood brain barrier (BBB) has been described in more than 40% of brains with prion disease. With loss of BBB integrity, it is expected that dying endothelial cells in the intracranial capillary vascular system, and specially dying neurons, can release PrP to the blood circulation system. Increased levels of plasma t-PrP have been reported in sporadic, genetic and variant CJD. However, increase in plasma t-PrP is not specific to prion diseases because it has also been reported in other conditions, such as AD, FTD, or LBD [96]. Therefore, it seems to be useful to discriminate between neurodegenerative and non-neurodegenerative dementias, but less clearly between prion diseases and other neurodegenerative disorders, even though higher levels have been reported in classical sCJD compared to other neurodegenerative dementias [96]. Interestingly, there is a dissociation between t-PrP CSF and plasma levels in sCJD, which are increased in plasma but decreased in CSF [62,96]. The presence of higher t-PrP levels in sCJD cases harboring MM and VV at PRNP codon 129 compared to MV carriers has also been reported, suggesting a better performance for homozygous carriers [88]. A mild association has been reported between CSF markers of neuronal injury (14-3-3 protein and NfL) and plasmatic t-PrP, suggesting that plasma t-PrP might be regarded as a biomarker of neurodegeneration, in contrast to CSF t-PrP, which probably reflects pathogenic PrP aggregation occurring in prion disease patients. Non-association has been reported between plasma t-PrP and the stage or disease duration, which argues against a potential use of this marker for prognostic purposes [96]. Advantages: It is possible to assess this biomarker in an accessible fluid, so invasive techniques are not required. The increase in t-PrP in the plasma of CJD (sporadic, acquired and genetic) can be detected from the early stages and does not vary between different stages of the disease, making it potentially useful as an early diagnosis biomarker. Limitations: It is not specific for CJD diagnosis, and we do not have information about less prevalent prionopathies. This biomarker seems to be more related to neurodegeneration. Medicina 2022, 58, 473 13 of 19 7. Nasal Mucosa RT-QuIC performed in the samples obtained by nasal brushing, a less invasive tech- nique compared to lumbar puncture, has shown a very high diagnosis accuracy according to preliminary data, with a sensitivity of 97% and specificity of 100%; this is a better perfor- mance compared to RT-QuIC in CSF [97,98]. It has been suggested that the combination of CSF RT-QuIC and RT-QuIC from a sample of nasal mucosa could increase the sensitivity in the early diagnosis of CJD to almost 100% [42,61]. Advantages: Obtaining a nasal mucosal sample would be safer and better tolerated by patients than lumbar puncture. If the preliminary data are confirmed, it could potentially replace CSF as a sample for RT-QuIC, or, at least, and this is ultimately more likely, allow increased diagnostic confidence in those subjects with high suspicion of prion disease with negative RT-QuIC in CSF, with the ability to detect some false negatives. Limitations: Only preliminary data are available that do not allow evaluation of its practical clinical applicability. In many subjects, lumbar puncture will continue to be performed for screening of other neurodegenerative dementias; therefore, it seems implausible that nasal mucosa sampling can universally replace lumbar puncture for RT- QuIC study, at least not until other biomarkers with good diagnosis performance for other neurodegenerative dementias are developed for use in nasal mucosa. 8. Future Directions The combination of neuroimaging (structural magnetic resonance) and CSF RT-QuIC for prion protein, t-tau, t-tau/p-tau ratio and 14-3-3 protein appears to be the most cost- effective combination for the early diagnosis of different prion diseases. Now that t-tau and t-tau/p-tau ratio are frequently assessed in mild cognitive impairment, considering whether abnormally high levels of one or both are highly suggestive of prion disease is essential. Qualitative analysis of EEG seems to be less useful for early diagnosis. However, research in the applicability of quantitative EEG to detect changes from earlier stages of the disease (including presymptomatic) could substantially increase its diagnostic performance, as well as reduce interobserver variability. PSG continues to be essential for the diagnosis of sFI and FFI, and review of EEG during PSG could potentially increase the capacity to detect PSWCs in CJD patients. Future studies to assess the performance of routine EEG vs. PSG-EEG (qualitative and quantitative analyses) would be of interest. The performance of CSF biomarkers is very good, but their assessment still requires an invasive test (lumbar puncture) that is usually performed later after symptom onset. The identification of diagnostic biomarkers in more accessible fluids, such as peripheral blood, is currently being investigated. The most promising plasma biomarkers are very sensitive and non-specific (NfL and t-tau) and require ultra-sensitive analysis techniques that are unavailable in most centers. Nevertheless, despite technical limitations, peripheral blood is the best potential source for future biomarkers that, ideally, should also directly reflect prion pathology. PrPSc measured in exosomes, small extracellular vesicles capable of crossing the blood-brain barrier, in both peripheral blood and CSF, are being suggested as possible biomarkers for the accurate diagnosis of prion diseases, but validation studies are still lacking [99,100]. Even if the novel strategy of PrPSc determination in exosomes in peripheral blood fails, efforts to identify other biomarkers with the same, or at least very close, performance to RT-QuIC in CSF, the best available diagnostic biomarker, in more accessible fluids, such as peripheral blood and/or mucosa, will remain a priority. Genetic prion diseases, on the other hand, are not only candidates for the application of potential future disease-modifying treatments (such as antisense oligonucleotides) but also allow the identification of prognostic and/or disease progression biomarkers. Confirming that periodic plasma NfL determination can predict up to two years in advance the onset of symptoms [90], and that higher t-tau levels are associated with a faster disease course, could help to better select potential candidates for future clinical trials. The availability of objective biomarkers of progression is essential to consider how to measure response in future clinical trials, as these are pathologies with widely varying clinical phenotypes Medicina 2022, 58, 473 14 of 19 and age of symptom onset [101]. Therefore, the identification of accessible prognostic biomarkers to be measured repeatedly in the population at risk is an undeniable need. 9. Conclusions The early and accurate diagnosis of prion diseases with a clinically progressive course and without modifying treatment is essential. Therefore, the availability of biomarkers that can provide near-total diagnostic certainty is crucial. The fact that some of these biomarkers are found in high concentrations from an early stage (even in asymptomatic carriers in the genetic forms) can help us to better identify subjects that could be candidates for future therapeutic strategies. Among the biomarkers available in clinical practice, RT-QuIC in cerebrospinal fluid, is the diagnostic biomarker with the best performance for early diagnosis; preliminary results of the same technique in other tissues, such as nasal mucosa, are also very promising. However, along with the determination of RT-QuIC, it is advisable to also request the determination of 14-3-3 protein, t-tau and p-tau, since diagnostic sensitivity will be further increased by their use without any decrease in specificity. In recent years, there have been fewer advances in neuroimaging and neurophysiology biomarkers, but they are no less relevant. Structural neuroimaging (MR should be used preferably) within the etiological study of any neurodegenerative dementia is mandatory and will therefore continue to have a relevant role. Neurophysiological tests will continue to play an essential role in the diagnosis of sporadic or fatal familial insomnia, and the electroencephalogram, which is non-invasive and inexpensive, can remain as an additional diagnostic tool in centers with fewer resources. For the time being, it does not seem that plasma biomarkers will be applied for the diagnosis of prion diseases due to the inferior performance of all biomarkers studied in the blood in comparison to cerebrospinal fluid to discriminate prion diseases from other neurodegenerative dementias. However, they could be useful for monitoring the response to a possible disease-modifying treatment, since assay protocols based on peripheral blood biomarkers as a target response will always be easier to perform than those based on CSF biomarkers. Author Contributions: Conceptualization, M.A.; methodology, M.A. and I.R.; investigation, M.A. and I.R.; resources, M.A.; data curation, M.A. and I.R.; writing—original draft preparation, M.A. and I.R.; writing—review and editing, M.A., M.V.Z. and M.M.; visualization, M.A., M.V.Z. and M.M.; supervision, M.A., M.V.Z. and M.M.; project administration, M.A.; funding acquisition, M.A. All authors have read and agreed to the published version of the manuscript. Funding: MA acknowledges support from a Río Hortega Fellowship (CM19/00066) by the Carlos III Health Institute. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Olivia Belbin, from the Memory Unit of Hospital Santa Creu i Sant Pau, has provided the license of Biorender.com to include one of the figures in this paper. Conflicts of Interest: The authors declare no conflict of interest. References 1. Llorens, F.; Rübsamen, N.; Hermann, P.; Schmitz, M.; Villar-Piqué, A.; Goebel, S.; Karch, A.; Zerr, I. A Prognostic Model for Overall Survival in Sporadic Creutzfeldt-Jakob Disease. Alzheimer’s Dement. 2020, 16, 1438–1447. [CrossRef] [PubMed] 2. Ladogana, A.; Kovacs, G.G. Genetic Creutzfeldt-Jakob Disease. Handb. Clin. Neurol. 2018, 153, 219–242. [CrossRef] [PubMed] 3. Hermann, P.; Appleby, B.; Brandel, J.-P.; Caughey, B.; Collins, S.; Geschwind, M.D.; Green, A.; Haïk, S.; Kovacs, G.G.; Ladogana, A.; et al. Biomarkers and Diagnostic Guidelines for Sporadic Creutzfeldt-Jakob Disease. Lancet Neurol. 2021, 20, 235–246. [CrossRef] Medicina 2022, 58, 473 15 of 19 4. Atkinson, C.J.; Zhang, K.; Munn, A.L.; Wiegmans, A.; Wei, M.Q. Prion Protein Scrapie and the Normal Cellular Prion Protein. Prion 2016, 10, 63–82. [CrossRef] 5. Blennow, K.; Diaz-Lucena, D.; Zetterberg, H.; Villar-Pique, A.; Karch, A.; Vidal, E.; Hermann, P.; Schmitz, M.; Ferrer Abizanda, I.; Zerr, I.; et al. CSF Neurogranin as a Neuronal Damage Marker in CJD: A Comparative Study with AD. J. Neurol. Neurosurg. Psychiatry 2019, 90, 846–853. [CrossRef] 6. Marino, S.; Morabito, R.; De Salvo, S.; Bonanno, L.; Bramanti, A.; Pollicino, P.; Giorgianni, R.; Bramanti, P. Quantitative, Functional MRI and Neurophysiological Markers in a Case of Gerstmann-Sträussler-Scheinker Syndrome. Funct. Neurol. 2017, 32, 153–158. [CrossRef] [PubMed] 7. Moore, R.C.; Xiang, F.; Monaghan, J.; Han, D.; Zhang, Z.; Edström, L.; Anvret, M.; Prusiner, S.B. Huntington Disease Phenocopy Is a Familial Prion Disease. Am. J. Hum. Genet. 2001, 69, 1385–1388. [CrossRef] [PubMed] 8. Brandel, J.-P.; Knight, R. Variant Creutzfeldt-Jakob Disease. Handb. Clin. Neurol. 2018, 153, 191–205. [CrossRef] [PubMed] 9. Spencer, M.D.; Knight, R.S.G.; Will, R.G. First Hundred Cases of Variant Creutzfeldt-Jakob Disease: Retrospective Case Note Review of Early Psychiatric and Neurological Features. BMJ 2002, 324, 1479–1482. [CrossRef] 10. Mok, T.H.; Mead, S. Preclinical Biomarkers of Prion Infection and Neurodegeneration. Curr. Opin. Neurobiol. 2020, 61, 82–88. [CrossRef] 11. Hill, A.F.; Joiner, S.; Wadsworth, J.D.F.; Sidle, K.C.L.; Bell, J.E.; Budka, H.; Ironside, J.W.; Collinge, J. Molecular Classification of Sporadic Creutzfeldt–Jakob Disease. Brain 2003, 126, 1333–1346. [CrossRef] [PubMed] 12. Safar, J.G. Molecular Pathogenesis of Sporadic Prion Diseases in Man. Prion 2012, 6, 108–115. [CrossRef] [PubMed] 13. Ascari, L.M.; Rocha, S.C.; Gonçalves, P.B.; Vieira, T.C.R.G.; Cordeiro, Y. Challenges and Advances in Antemortem Diagnosis of Human Transmissible Spongiform Encephalopathies. Front. Bioeng. Biotechnol. 2020, 8, 585896. [CrossRef] [PubMed] 14. Abu-Rumeileh, S.; Baiardi, S.; Polischi, B.; Mammana, A.; Franceschini, A.; Green, A.; Capellari, S.; Parchi, P. Diagnostic Value of Surrogate CSF Biomarkers for Creutzfeldt-Jakob Disease in the Era of RT-QuIC. J. Neurol. 2019, 266, 3136–3143. [CrossRef] [PubMed] 15. Wieser, H.G.; Schindler, K.; Zumsteg, D. EEG in Creutzfeldt-Jakob Disease. Clin. Neurophysiol. Off. J. Int. Fed. Clin. Neurophysiol. 2006, 117, 935–951. [CrossRef] 16. Skillbäck, T.; Rosén, C.; Asztely, F.; Mattsson, N.; Blennow, K.; Zetterberg, H. Diagnostic Performance of Cerebrospinal Fluid Total Tau and Phosphorylated Tau in Creutzfeldt-Jakob Disease: Results from the Swedish Mortality Registry. JAMA Neurol. 2014, 71, 476–483. [CrossRef] 17. Figgie, M.P.J.; Appleby, B.S. Clinical Use of Improved Diagnostic Testing for Detection of Prion Disease. Viruses 2021, 13, 789. [CrossRef] [PubMed] 18. Franko, E.; Wehner, T.; Joly, O.; Lowe, J.; Porter, M.-C.; Kenny, J.; Thompson, A.; Rudge, P.; Collinge, J.; Mead, S. Quantitative EEG Parameters Correlate with the Progression of Human Prion Diseases. J. Neurol. Neurosurg. Psychiatry 2016, 87, 1061–1067. [CrossRef] 19. Matsubayashi, T.; Akaza, M.; Hayashi, Y.; Hamaguchi, T.; Yamada, M.; Shimohata, T.; Yokota, T.; Sanjo, N. Focal Sharp Waves Are a Specific Early-Stage Marker of the MM2-Cortical Form of Sporadic Creutzfeldt-Jakob Disease. Prion 2020, 14, 207–213. [CrossRef] 20. Wu, L.-Y.; Zhan, S.-Q.; Huang, Z.-Y.; Zhang, B.; Wang, T.; Liu, C.-F.; Lu, H.; Dong, X.-P.; Wu, Z.-Y.; Zhang, J.-W.; et al. Expert Consensus on Clinical Diagnostic Criteria for Fatal Familial Insomnia. Chin. Med. J. 2018, 131, 1613–1617. [CrossRef] 21. Kang, P.; de Bruin, G.S.; Wang, L.H.; Ward, B.A.; Ances, B.M.; Lim, M.M.; Bucelli, R.C. Sleep Pathology in Creutzfeldt-Jakob Disease. J. Clin. Sleep Med. JCSM Off. Publ. Am. Acad. Sleep Med. 2016, 12, 1033–1039. [CrossRef] [PubMed] 22. Dai, Y.; Shao, J.; Lang, Y.; Lv, Y.; Cui, L. Clinical Manifestations and Polysomnography-Based Analysis in Nine Cases of Probable Sporadic Creutzfeldt-Jakob Disease. Neurol. Sci. Off. J. Ital. Neurol. Soc. Ital. Soc. Clin. Neurophysiol. 2021, 42, 4209–4219. [CrossRef] [PubMed] 23. Macfarlane, R.G.; Wroe, S.J.; Collinge, J.; Yousry, T.A.; Jäger, H.R. Neuroimaging Findings in Human Prion Disease. J. Neurol. Neurosurg. Psychiatry 2007, 78, 664–670. [CrossRef] [PubMed] 24. Sacco, S.; Paoletti, M.; Staffaroni, A.M.; Kang, H.; Rojas, J.; Marx, G.; Goh, S.-Y.; Luisa Mandelli, M.; Allen, I.E.; Kramer, J.H.; et al. Multimodal MRI Staging for Tracking Progression and Clinical-Imaging Correlation in Sporadic Creutzfeldt-Jakob Disease. NeuroImage. Clin. 2021, 30, 102523. [CrossRef] [PubMed] 25. Baiardi, S.; Magherini, A.; Capellari, S.; Redaelli, V.; Ladogana, A.; Rossi, M.; Tagliavini, F.; Pocchiari, M.; Giaccone, G.; Parchi, P. Towards an Early Clinical Diagnosis of Sporadic CJD VV2 (Ataxic Type). J. Neurol. Neurosurg. Psychiatry 2017, 88, 764–772. [CrossRef] 26. Ioannides, P.; Karacostas, D. Neuroimaging in Human Prion Disease: Searching in the Mist. World J. Radiol. 2009, 1, 45–49. [CrossRef] 27. Divya, K.P.; Menon, R.N.; Thomas, B.; Nair, M. A Hospital-Based Registry of Creutzfeldt-Jakob Disease: Can Neuroimaging Serve as a Surrogate Biomarker? Neurol. India 2016, 64, 411–418. [CrossRef] 28. Rudge, P.; Hyare, H.; Green, A.; Collinge, J.; Mead, S. Imaging and CSF Analyses Effectively Distinguish CJD from Its Mimics. J. Neurol. Neurosurg. Psychiatry 2018, 89, 461–466. [CrossRef] 29. Kim, J.H.; Choi, B.S.; Jung, C.; Chang, Y.; Kim, S. Diffusion-Weighted Imaging and Magnetic Resonance Spectroscopy of Sporadic Creutzfeldt-Jakob Disease: Correlation with Clinical Course. Neuroradiology 2011, 53, 939–945. [CrossRef] Medicina 2022, 58, 473 16 of 19 30. Shiga, Y.; Miyazawa, K.; Sato, S.; Fukushima, R.; Shibuya, S.; Sato, Y.; Konno, H.; Doh-ura, K.; Mugikura, S.; Tamura, H.; et al. Diffusion-Weighted MRI Abnormalities as an Early Diagnostic Marker for Creutzfeldt-Jakob Disease. Neurology 2004, 63, 443–449. [CrossRef] 31. Lee, H.; Rosenmann, H.; Chapman, J.; Kingsley, P.B.; Hoffmann, C.; Cohen, O.S.; Kahana, E.; Korczyn, A.D.; Prohovnik, I. Thalamo-Striatal Diffusion Reductions Precede Disease Onset in Prion Mutation Carriers. Brain 2009, 132, 2680–2687. [CrossRef] [PubMed] 32. Kim, E.-J.; Cho, S.-S.; Jeong, B.-H.; Kim, Y.-S.; Seo, S.W.; Na, D.L.; Geschwind, M.D.; Jeong, Y. Glucose Metabolism in Sporadic Creutzfeldt-Jakob Disease: A Statistical Parametric Mapping Analysis of (18) F-FDG PET. Eur. J. Neurol. 2012, 19, 488–493. [CrossRef] [PubMed] 33. Green, A.J.E.; Ramljak, S.; Müller, W.E.G.; Knight, R.S.G.; Schröder, H.C. 14-3-3 in the Cerebrospinal Fluid of Patients with Variant and Sporadic Creutzfeldt-Jakob Disease Measured Using Capture Assay Able to Detect Low Levels of 14-3-3 Protein. Neurosci. Lett. 2002, 324, 57–60. [CrossRef] 34. Matsui, Y.; Satoh, K.; Miyazaki, T.; Shirabe, S.; Atarashi, R.; Mutsukura, K.; Satoh, A.; Kataoka, Y.; Nishida, N. High Sensitivity of an ELISA Kit for Detection of the Gamma-Isoform of 14-3-3 Proteins: Usefulness in Laboratory Diagnosis of Human Prion Disease. BMC Neurol. 2011, 11, 120. [CrossRef] [PubMed] 35. Castellani, R.J.; Colucci, M.; Xie, Z.; Zou, W.; Li, C.; Parchi, P.; Capellari, S.; Pastore, M.; Rahbar, M.H.; Chen, S.G.; et al. Sensitivity of 14-3-3 Protein Test Varies in Subtypes of Sporadic Creutzfeldt-Jakob Disease. Neurology 2004, 63, 436–442. [CrossRef] [PubMed] 36. Leitão, M.J.; Baldeiras, I.; Almeida, M.R.; Ribeiro, M.H.; Santos, A.C.; Ribeiro, M.; Tomás, J.; Rocha, S.; Santana, I.; Oliveira, C.R. CSF Tau Proteins Reduce Misdiagnosis of Sporadic Creutzfeldt-Jakob Disease Suspected Cases with Inconclusive 14-3-3 Result. J. Neurol. 2016, 263, 1847–1861. [CrossRef] [PubMed] 37. Torres, M.; Cartier, L.; Matamala, J.M.; Hernández, N.; Woehlbier, U.; Hetz, C. Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease. PLoS ONE 2012, 7, e36159. [CrossRef] 38. Ladogana, A.; Sanchez-Juan, P.; Mitrová, E.; Green, A.; Cuadrado-Corrales, N.; Sánchez-Valle, R.; Koscova, S.; Aguzzi, A.; Sklaviadis, T.; Kulczycki, J.; et al. Cerebrospinal Fluid Biomarkers in Human Genetic Transmissible Spongiform Encephalopathies. J. Neurol. 2009, 256, 1620–1628. [CrossRef] 39. Higuma, M.; Sanjo, N.; Satoh, K.; Shiga, Y.; Sakai, K.; Nozaki, I.; Hamaguchi, T.; Nakamura, Y.; Kitamoto, T.; Shirabe, S.; et al. Relationships between Clinicopathological Features and Cerebrospinal Fluid Biomarkers in Japanese Patients with Genetic Prion Diseases. PLoS ONE 2013, 8, e60003. [CrossRef] 40. Leitão, M.J.; Baldeiras, I.; Almeida, M.R.; Ribeiro, M.H.; Santos, A.C.; Ribeiro, M.; Tomás, J.; Rocha, S.; Santana, I.; Oliveira, C.R. Sporadic Creutzfeldt-Jakob Disease Diagnostic Accuracy Is Improved by a New CSF ELISA 14-3-3γ Assay. Neuroscience 2016, 322, 398–407. [CrossRef] 41. Lattanzio, F.; Abu-Rumeileh, S.; Franceschini, A.; Kai, H.; Amore, G.; Poggiolini, I.; Rossi, M.; Baiardi, S.; McGuire, L.; Ladogana, A.; et al. Prion-Specific and Surrogate CSF Biomarkers in Creutzfeldt-Jakob Disease: Diagnostic Accuracy in Re- lation to Molecular Subtypes and Analysis of Neuropathological Correlates of p-Tau and Aβ42 Levels. Acta Neuropathol. 2017, 133, 559–578. [CrossRef] [PubMed] 42. Zanusso, G.; Fiorini, M.; Ferrari, S.; Gajofatto, A.; Cagnin, A.; Galassi, A.; Richelli, S.; Monaco, S. Cerebrospinal Fluid Markers in Sporadic Creutzfeldt-Jakob Disease. Int. J. Mol. Sci. 2011, 12, 6281–6292. [CrossRef] [PubMed] 43. Hyeon, J.W.; Kim, S.Y.; Lee, J.; Park, J.S.; Hwang, K.J.; Lee, S.M.; An, S.A.; Lee, M.K.; Ju, Y.R. Alternative Application of Tau Protein in Creutzfeldt-Jakob Disease Diagnosis: Improvement for Weakly Positive 14-3-3 Protein in the Laboratory. Sci. Rep. 2015, 5, 15283. [CrossRef] [PubMed] 44. Chohan, G.; Pennington, C.; Mackenzie, J.M.; Andrews, M.; Everington, D.; Will, R.G.; Knight, R.S.G.; Green, A.J.E. The Role of Cerebrospinal Fluid 14-3-3 and Other Proteins in the Diagnosis of Sporadic Creutzfeldt-Jakob Disease in the UK: A 10-Year Review. J. Neurol. Neurosurg. Psychiatry 2010, 81, 1243–1248. [CrossRef] [PubMed] 45. Lemstra, A.W.; van Meegen, M.T.; Vreyling, J.P.; Meijerink, P.H.; Jansen, G.H.; Bulk, S.; Baas, F.; van Gool, W.A. 14-3-3 Testing in Diagnosing Creutzfeldt-Jakob Disease: A Prospective Study in 112 Patients. Neurology 2000, 55, 514–516. [CrossRef] 46. Schmitz, M.; Ebert, E.; Stoeck, K.; Karch, A.; Collins, S.; Calero, M.; Sklaviadis, T.; Laplanche, J.-L.; Golanska, E.; Baldeiras, I.; et al. Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic. Mol. Neurobiol. 2016, 53, 2189–2199. [CrossRef] 47. Stoeck, K.; Sanchez-Juan, P.; Gawinecka, J.; Green, A.; Ladogana, A.; Pocchiari, M.; Sanchez-Valle, R.; Mitrova, E.; Sklaviadis, T.; Kulczycki, J.; et al. Cerebrospinal Fluid Biomarker Supported Diagnosis of Creutzfeldt-Jakob Disease and Rapid Dementias: A Longitudinal Multicentre Study over 10 Years. Brain 2012, 135, 3051–3061. [CrossRef] 48. Llorens, F.; Karch, A.; Golanska, E.; Schmitz, M.; Lange, P.; Sikorska, B.; Liberski, P.P.; Zerr, I. Cerebrospinal Fluid Biomarker-Based Diagnosis of Sporadic Creutzfeldt-Jakob Disease: A Validation Study for Previously Established Cutoffs. Dement. Geriatr. Cogn. Disord. 2017, 43, 71–80. [CrossRef] 49. Hamlin, C.; Puoti, G.; Berri, S.; Sting, E.; Harris, C.; Cohen, M.; Spear, C.; Bizzi, A.; Debanne, S.M.; Rowland, D.Y. A Comparison of Tau and 14-3-3 Protein in the Diagnosis of Creutzfeldt-Jakob Disease. Neurology 2012, 79, 547–552. [CrossRef] 50. Coulthart, M.B.; Jansen, G.H.; Olsen, E.; Godal, D.L.; Connolly, T.; Choi, B.C.K.; Wang, Z.; Cashman, N.R. Diagnostic Accuracy of Cerebrospinal Fluid Protein Markers for Sporadic Creutzfeldt-Jakob Disease in Canada: A 6-Year Prospective Study. BMC Neurol. 2011, 11, 133. [CrossRef] Medicina 2022, 58, 473 17 of 19 51. Foucault-Fruchard, L.; Delaye, J.B.; Morange, V.; Beaufils, E.; Duwicquet, C.; Quadrio, I.; Balageas, A.C.; Dufour-Rainfray, D. An Automated Alert System Based on the P-Tau/Tau Ratio to Quickly Inform Health Professionals upon a Suspected Case of Sporadic Creutzfeldt-Jakob Disease. J. Neurol. Sci. 2020, 415, 116971. [CrossRef] [PubMed] 52. Llorens, F.; Villar-Piqué, A.; Hermann, P.; Schmitz, M.; Goebel, S.; Waniek, K.; Lachmann, I.; Zerr, I. Cerebrospinal Fluid Non- Phosphorylated Tau in the Differential Diagnosis of Creutzfeldt-Jakob Disease: A Comparative Prospective Study with 14-3-3. J. Neurol. 2020, 267, 543–550. [CrossRef] [PubMed] 53. Meiner, Z.; Kahana, E.; Baitcher, F.; Korczyn, A.D.; Chapman, J.; Cohen, O.S.; Milo, R.; Aharon-Perez, J.; Abramsky, O.; Gabizon, R.; et al. Tau and 14-3-3 of Genetic and Sporadic Creutzfeldt-Jakob Disease Patients in Israel. J. Neurol. 2011, 258, 255–262. [CrossRef] 54. Gmitterová, K.; Heinemann, U.; Krasnianski, A.; Gawinecka, J.; Zerr, I. Cerebrospinal Fluid Markers in the Differentiation of Molecular Subtypes of Sporadic Creutzfeldt-Jakob Disease. Eur. J. Neurol. 2016, 23, 1126–1133. [CrossRef] [PubMed] 55. Karch, A.; Hermann, P.; Ponto, C.; Schmitz, M.; Arora, A.; Zafar, S.; Llorens, F.; Müller-Heine, A.; Zerr, I. Cerebrospinal Fluid Tau Levels Are a Marker for Molecular Subtype in Sporadic Creutzfeldt-Jakob Disease. Neurobiol. Aging 2015, 36, 1964–1968. [CrossRef] [PubMed] 56. Karch, A.; Llorens, F.; Schmitz, M.; Arora, A.S.; Zafar, S.; Lange, P.; Schmidt, C.; Zerr, I. Stratification by Genetic and Demographic Characteristics Improves Diagnostic Accuracy of Cerebrospinal Fluid Biomarkers in Rapidly Progressive Dementia. J. Alzheimer’s Dis. 2016, 54, 1385–1393. [CrossRef] 57. Satoh, K.; Shirabe, S.; Eguchi, H.; Tsujino, A.; Motomura, M.; Satoh, A.; Tsujihata, M.; Eguchi, K. Chronological Changes in MRI and CSF Biochemical Markers in Creutzfeldt-Jakob Disease Patients. Dement. Geriatr. Cogn. Disord. 2007, 23, 372–381. [CrossRef] 58. Cohen, O.S.; Chapman, J.; Korczyn, A.D.; Siaw, O.L.; Warman-Alaluf, N.; Nitsan, Z.; Appel, S.; Kahana, E.; Rosenmann, H.; Hoffmann, C. CSF Tau Correlates with the Degree of Cortical Involvement in E200K Familial Creutzfeldt-Jakob Disease. Neurosci. Lett. 2016, 634, 76–78. [CrossRef] 59. Van Everbroeck, B.; Quoilin, S.; Boons, J.; Martin, J.J.; Cras, P. A Prospective Study of CSF Markers in 250 Patients with Possible Creutzfeldt-Jakob Disease. J. Neurol. Neurosurg. Psychiatry 2003, 74, 1210–1214. [CrossRef] 60. Wang, G.-R.; Gao, C.; Shi, Q.; Zhou, W.; Chen, J.-M.; Dong, C.-F.; Shi, S.; Wang, X.; Wei, Y.; Jiang, H.-Y.; et al. Elevated Levels of Tau Protein in Cerebrospinal Fluid of Patients with Probable Creutzfeldt-Jakob Disease. Am. J. Med. Sci. 2010, 340, 291–295. [CrossRef] 61. Thompson, A.G.B.; Mead, S.H. Review: Fluid Biomarkers in the Human Prion Diseases. Mol. Cell. Neurosci. 2019, 97, 81–92. [CrossRef] 62. Zerr, I.; Schmitz, M.; Karch, A.; Villar-Piqué, A.; Kanata, E.; Golanska, E.; Díaz-Lucena, D.; Karsanidou, A.; Hermann, P.; Knipper, T.; et al. Cerebrospinal Fluid Neurofilament Light Levels in Neurodegenerative Dementia: Evaluation of Diagnostic Accuracy in the Differential Diagnosis of Prion Diseases. Alzheimer’s Dement. 2018, 14, 751–763. [CrossRef] [PubMed] 63. Kanata, E.; Golanska, E.; Villar-Piqué, A.; Karsanidou, A.; Dafou, D.; Xanthopoulos, K.; Schmitz, M.; Ferrer, I.; Karch, A.; Sikorska, B.; et al. Cerebrospinal Fluid Neurofilament Light in Suspected Sporadic Creutzfeldt-Jakob Disease. J. Clin. Neurosci. Off. J. Neurosurg. Soc. Australas. 2019, 60, 124–127. [CrossRef] [PubMed] 64. Abu-Rumeileh, S.; Capellari, S.; Stanzani-Maserati, M.; Polischi, B.; Martinelli, P.; Caroppo, P.; Ladogana, A.; Parchi, P. The CSF Neurofilament Light Signature in Rapidly Progressive Neurodegenerative Dementias. Alzheimer’s Res. Ther. 2018, 10, 3. [CrossRef] [PubMed] 65. Llorens, F.; Kruse, N.; Schmitz, M.; Shafiq, M.; da Cunha, J.E.G.; Gotzman, N.; Zafar, S.; Thune, K.; de Oliveira, J.R.M.; Mollenhauer, B.; et al. Quantification of CSF Biomarkers Using an Electrochemiluminescence-Based Detection System in the Differential Diagnosis of AD and SCJD. J. Neurol. 2015, 262, 2305–2311. [CrossRef] [PubMed] 66. Llorens, F.; Kruse, N.; Schmitz, M.; Gotzmann, N.; Golanska, E.; Thüne, K.; Zejneli, O.; Kanata, E.; Knipper, T.; Cramm, M.; et al. Evaluation of α-Synuclein as a Novel Cerebrospinal Fluid Biomarker in Different Forms of Prion Diseases. Alzheimer’s Dement. 2017, 13, 710–719. [CrossRef] [PubMed] 67. Oeckl, P.; Metzger, F.; Nagl, M.; von Arnim, C.A.F.; Halbgebauer, S.; Steinacker, P.; Ludolph, A.C.; Otto, M. Alpha-, Beta-, and Gamma-Synuclein Quantification in Cerebrospinal Fluid by Multiple Reaction Monitoring Reveals Increased Concentrations in Alzheimer’s and Creutzfeldt-Jakob Disease but No Alteration in Synucleinopathies. Mol. Cell. Proteom. 2016, 15, 3126–3138. [CrossRef] [PubMed] 68. Mastrangelo, A.; Baiardi, S.; Zenesini, C.; Poleggi, A.; Mammana, A.; Polischi, B.; Ladogana, A.; Capellari, S.; Parchi, P. Diagnostic and Prognostic Performance of CSF α-Synuclein in Prion Disease in the Context of Rapidly Progressive Dementia. Alzheimer’s Dement. 2021, 13, e12214. [CrossRef] 69. Abu-Rumeileh, S.; Oeckl, P.; Baiardi, S.; Halbgebauer, S.; Steinacker, P.; Capellari, S.; Otto, M.; Parchi, P. CSF Ubiquitin Levels Are Higher in Alzheimer’s Disease than in Frontotemporal Dementia and Reflect the Molecular Subtype in Prion Disease. Biomolecules 2020, 10, 497. [CrossRef] 70. Steinacker, P.; Rist, W.; Swiatek-de-Lange, M.; Lehnert, S.; Jesse, S.; Pabst, A.; Tumani, H.; von Arnim, C.A.F.; Mitrova, E.; Kretzschmar, H.A.; et al. Ubiquitin as Potential Cerebrospinal Fluid Marker of Creutzfeldt-Jakob Disease. Proteomics 2010, 10, 81–89. [CrossRef] 71. Chen, C.; Hu, C.; Zhou, W.; Chen, J.; Shi, Q.; Xiao, K.; Wang, Y.; Dong, X.-P. Calmodulin Level Is Significantly Increased in the Cerebrospinal Fluid of Patients with Sporadic Creutzfeldt-Jakob Disease. Eur. J. Neurol. 2021, 28, 1134–1141. [CrossRef] [PubMed] Medicina 2022, 58, 473 18 of 19 72. Schmitz, M.; Llorens, F.; Pracht, A.; Thom, T.; Correia, Â.; Zafar, S.; Ferrer, I.; Zerr, I. Regulation of Human Cerebrospinal Fluid Malate Dehydrogenase 1 in Sporadic Creutzfeldt-Jakob Disease Patients. Aging 2016, 8, 2927–2935. [CrossRef] [PubMed] 73. Zerr, I.; Villar-Piqué, A.; Schmitz, V.E.; Poleggi, A.; Pocchiari, M.; Sánchez-Valle, R.; Calero, M.; Calero, O.; Baldeiras, I.; Santana, I.; et al. Evaluation of Human Cerebrospinal Fluid Malate Dehydrogenase 1 as a Marker in Genetic Prion Disease Patients. Biomolecules 2019, 9, 800. [CrossRef] [PubMed] 74. Abu-Rumeileh, S.; Steinacker, P.; Polischi, B.; Mammana, A.; Bartoletti-Stella, A.; Oeckl, P.; Baiardi, S.; Zenesini, C.; Huss, A.; Cortelli, P.; et al. CSF Biomarkers of Neuroinflammation in Distinct Forms and Subtypes of Neurodegenerative Dementia. Alzheimer’s Res. Ther. 2019, 12, 2. [CrossRef] [PubMed] 75. Herbst, A.; McIlwain, S.; Schmidt, J.J.; Aiken, J.M.; Page, C.D.; Li, L. Prion Disease Diagnosis by Proteomic Profiling. J. Proteome Res. 2009, 8, 1030–1036. [CrossRef] 76. Foutz, A.; Appleby, B.S.; Hamlin, C.; Liu, X.; Yang, S.; Cohen, Y.; Chen, W.; Blevins, J.; Fausett, C.; Wang, H.; et al. Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid. Ann. Neurol. 2017, 81, 79–92. [CrossRef] 77. Sano, K.; Satoh, K.; Atarashi, R.; Takashima, H.; Iwasaki, Y.; Yoshida, M.; Sanjo, N.; Murai, H.; Mizusawa, H.; Schmitz, M.; et al. Early Detection of Abnormal Prion Protein in Genetic Human Prion Diseases Now Possible Using Real-Time QUIC Assay. PLoS ONE 2013, 8, e54915. [CrossRef] 78. Dorey, A.; Tholance, Y.; Vighetto, A.; Perret-Liaudet, A.; Lachman, I.; Krolak-Salmon, P.; Wagner, U.; Struyfs, H.; De Deyn, P.P.; El-Moualij, B.; et al. Association of Cerebrospinal Fluid Prion Protein Levels and the Distinction between Alzheimer Disease and Creutzfeldt-Jakob Disease. JAMA Neurol. 2015, 72, 267–275. [CrossRef] 79. Vallabh, S.M.; Minikel, E.V.; Williams, V.J.; Carlyle, B.C.; McManus, A.J.; Wennick, C.D.; Bolling, A.; Trombetta, B.A.; Urick, D.; Nobuhara, C.K.; et al. Cerebrospinal Fluid and Plasma Biomarkers in Individuals at Risk for Genetic Prion Disease. BMC Med. 2020, 18, 140. [CrossRef] 80. Villar-Piqué, A.; Schmitz, M.; Lachmann, I.; Karch, A.; Calero, O.; Stehmann, C.; Sarros, S.; Ladogana, A.; Poleggi, A.; Santana, I.; et al. Cerebrospinal Fluid Total Prion Protein in the Spectrum of Prion Diseases. Mol. Neurobiol. 2019, 56, 2811– 2821. [CrossRef] 81. Rhoads, D.D.; Wrona, A.; Foutz, A.; Blevins, J.; Glisic, K.; Person, M.; Maddox, R.A.; Belay, E.D.; Schonberger, L.B.; Tatsuoka, C.; et al. Diagnosis of Prion Diseases by RT-QuIC Results in Improved Surveillance. Neurology 2020, 95, e1017–e1026. [CrossRef] [PubMed] 82. Connor, A.; Wang, H.; Appleby, B.S.; Rhoads, D.D. Clinical Laboratory Tests Used to Aid in Diagnosis of Human Prion Disease. J. Clin. Microbiol. 2019, 57, e00769-19. [CrossRef] [PubMed] 83. Atarashi, R.; Satoh, K.; Sano, K.; Fuse, T.; Yamaguchi, N.; Ishibashi, D.; Matsubara, T.; Nakagaki, T.; Yamanaka, H.; Shirabe, S.; et al. Ultrasensitive Human Prion Detection in Cerebrospinal Fluid by Real-Time Quaking-Induced Conversion. Nat. Med. 2011, 17, 175–178. [CrossRef] [PubMed] 84. Piconi, G.; Peden, A.H.; Barria, M.A.; Green, A.J.E. Epitope Mapping of the Protease Resistant Products of RT-QuIC Does Not Allow the Discrimination of SCJD Subtypes. PLoS ONE 2019, 14, e0218509. [CrossRef] 85. Llorens, F.; Kruse, N.; Karch, A.; Schmitz, M.; Zafar, S.; Gotzmann, N.; Sun, T.; Köchy, S.; Knipper, T.; Cramm, M.; et al. Validation of α-Synuclein as a CSF Biomarker for Sporadic Creutzfeldt-Jakob Disease. Mol. Neurobiol. 2018, 55, 2249–2257. [CrossRef] 86. Orrù, C.D.; Groveman, B.R.; Hughson, A.G.; Manca, M.; Raymond, L.D.; Raymond, G.J.; Campbell, K.J.; Anson, K.J.; Kraus, A.; Caughey, B. RT-QuIC Assays for Prion Disease Detection and Diagnostics. Methods Mol. Biol. 2017, 1658, 185–203. [CrossRef] 87. Zerr, I.; Villar-Piqué, A.; Hermann, P.; Schmitz, M.; Varges, D.; Ferrer, I.; Riggert, J.; Zetterberg, H.; Blennow, K.; Llorens, F. Diagnostic and Prognostic Value of Plasma Neurofilament Light and Total-Tau in Sporadic Creutzfeldt-Jakob Disease. Alzheimer’s Res. Ther. 2021, 13, 86. [CrossRef] 88. Steinacker, P.; Blennow, K.; Halbgebauer, S.; Shi, S.; Ruf, V.; Oeckl, P.; Giese, A.; Kuhle, J.; Slivarichova, D.; Zetterberg, H.; et al. Neurofilaments in Blood and CSF for Diagnosis and Prediction of Onset in Creutzfeldt-Jakob Disease. Sci. Rep. 2016, 6, 38737. [CrossRef] 89. Abu-Rumeileh, S.; Baiardi, S.; Ladogana, A.; Zenesini, C.; Bartoletti-Stella, A.; Poleggi, A.; Mammana, A.; Polischi, B.; Pocchiari, M.; Capellari, S.; et al. Comparison between Plasma and Cerebrospinal Fluid Biomarkers for the Early Diagno- sis and Association with Survival in Prion Disease. J. Neurol. Neurosurg. Psychiatry 2020, 91, 1181–1188. [CrossRef] 90. Thompson, A.G.B.; Anastasiadis, P.; Druyeh, R.; Whitworth, I.; Nayak, A.; Nihat, A.; Mok, T.H.; Rudge, P.; Wadsworth, J.D.F.; Rohrer, J.; et al. Evaluation of Plasma Tau and Neurofilament Light Chain Biomarkers in a 12-Year Clinical Cohort of Human Prion Diseases. Mol. Psychiatry 2021, 26, 5955–5966. [CrossRef] 91. Abu-Rumeileh, S.; Parchi, P. Cerebrospinal Fluid and Blood Neurofilament Light Chain Protein in Prion Disease and Other Rapidly Progressive Dementias: Current State of the Art. Front. Neurosci. 2021, 15, 648743. [CrossRef] [PubMed] 92. Thompson, A.G.B.; Luk, C.; Heslegrave, A.J.; Zetterberg, H.; Mead, S.H.; Collinge, J.; Jackson, G.S. Neurofilament Light Chain and Tau Concentrations Are Markedly Increased in the Serum of Patients with Sporadic Creutzfeldt-Jakob Disease, and Tau Correlates with Rate of Disease Progression. J. Neurol. Neurosurg. Psychiatry 2018, 89, 955–961. [CrossRef] [PubMed] 93. Noguchi-Shinohara, M.; Hamaguchi, T.; Nozaki, I.; Sakai, K.; Yamada, M. Serum Tau Protein as a Marker for the Diagnosis of Creutzfeldt-Jakob Disease. J. Neurol. 2011, 258, 1464–1468. [CrossRef] [PubMed] 94. Villar-Piqué, A.; Schmitz, M.; Hermann, P.; Goebel, S.; Bunck, T.; Varges, D.; Ferrer, I.; Riggert, J.; Llorens, F.; Zerr, I. Plasma YKL-40 in the Spectrum of Neurodegenerative Dementia. J. Neuroinflamm. 2019, 16, 145. [CrossRef] Medicina 2022, 58, 473 19 of 19 95. Norsworthy, P.J.; Thompson, A.G.B.; Mok, T.H.; Guntoro, F.; Dabin, L.C.; Nihat, A.; Paterson, R.W.; Schott, J.M.; Collinge, J.; Mead, S.; et al. A Blood MiRNA Signature Associates with Sporadic Creutzfeldt-Jakob Disease Diagnosis. Nat. Commun. 2020, 11, 3960. [CrossRef] 96. Llorens, F.; Villar-Piqué, A.; Schmitz, M.; Diaz-Lucena, D.; Wohlhage, M.; Hermann, P.; Goebel, S.; Schmidt, I.; Glatzel, M.; Hauw, J.-J.; et al. Plasma Total Prion Protein as a Potential Biomarker for Neurodegenerative Dementia: Diagnostic Accuracy in the Spectrum of Prion Diseases. Neuropathol. Appl. Neurobiol. 2020, 46, 240–254. [CrossRef] 97. Behaeghe, O.; Mangelschots, E.; De Vil, B.; Cras, P. A Systematic Review Comparing the Diagnostic Value of 14-3-3 Protein in the Cerebrospinal Fluid, RT-QuIC and RT-QuIC on Nasal Brushing in Sporadic Creutzfeldt-Jakob Disease. Acta Neurol. Belg. 2018, 118, 395–403. [CrossRef] 98. Di Fede, G.; Giaccone, G.; Salmona, M.; Tagliavini, F. Translational Research in Alzheimer’s and Prion Diseases. J. Alzheimer’s Dis. 2018, 62, 1247–1259. [CrossRef] 99. López-Pérez, Ó.; Sanz-Rubio, D.; Hernaiz, A.; Betancor, M.; Otero, A.; Castilla, J.; Andréoletti, O.; Badiola, J.J.; Zaragoza, P.; Bolea, R.; et al. Cerebrospinal Fluid and Plasma Small Extracellular Vesicles and MiRNAs as Biomarkers for Prion Diseases. Int. J. Mol. Sci. 2021, 22, 6822. [CrossRef] 100. Vassileff, N.; Cheng, L.; Hill, A.F. Extracellular Vesicles—Propagators of Neuropathology and Sources of Potential Biomarkers and Therapeutics for Neurodegenerative Diseases. J. Cell Sci. 2020, 133. [CrossRef] 101. Minikel, E.V.; Vallabh, S.M.; Orseth, M.C.; Brandel, J.-P.; Haïk, S.; Laplanche, J.-L.; Zerr, I.; Parchi, P.; Capellari, S.; Safar, J.; et al. Age at Onset in Genetic Prion Disease and the Design of Preventive Clinical Trials. Neurology 2019, 93, e125–e134. [CrossRef] [PubMed]